Mutations Alter RNA-Mediated Conversion of Human Prions.
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ABSTRACT:
SUBMITTER: Alred EJ
PROVIDER: S-EPMC5928492 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The rate-limiting step in prion diseases is the initial transition of a prion protein from its native form into a mis-folded state in which the protein not only forms cell-toxic aggregates but also becomes infectious. Recent experiments implicate polyadenosine RNA as a possible agent for generating the initial seed. In order to understand the mechanism of RNA-mediated mis-folding and aggregation of prions, we dock polyadenosine RNA to mouse and human prion models. Changes in stability and secondary structure of the prions upon binding to polyadenosine RNA are evaluated by comparing molecular dynamics simulations of these complexes with that of the unbound prions.
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Project description:Sialylated glycans on the surface of mammalian cells act as part of a "self-associated molecular pattern," helping the immune system to recognize "self" from "altered self" or "nonself." To escape the host immune system, some bacterial pathogens have evolved biosynthetic pathways for host-like sialic acids, whereas others recruited host sialic acids for decorating their surfaces. Prions lack nucleic acids and are not conventional pathogens. Nevertheless, prions might use a similar strategy for invading and colonizing the lymphoreticular system. Here we show that the sialylation status of the infectious, disease-associated state of the prion protein (PrP(Sc)) changes with colonization of secondary lymphoid organs (SLOs). As a result, spleen-derived PrP(Sc) is more sialylated than brain-derived PrP(Sc). Enhanced sialylation of PrP(Sc) is recapitulated in vitro by incubating brain-derived PrP(Sc) with primary splenocytes or cultured macrophage RAW 264.7 cells. General inhibitors of sialyltranserases (STs), the enzymes that transfer sialic acid residues onto terminal positions of glycans, suppressed extrasialylation of PrP(Sc). A fluorescently labeled precursor of sialic acid revealed ST activity associated with RAW macrophages. This study illustrates that, upon colonization of SLOs, the sialylation status of prions changes by host STs. We propose that this mechanism is responsible for camouflaging prions in SLOs and has broad implications.
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Project description:High-throughput sequencing of hematologic malignancies and other cancers has revealed recurrent mis-sense mutations of genes encoding pre-mRNA splicing factors. The essential splicing factor U2AF2 recognizes a polypyrimidine-tract splice-site signal and initiates spliceosome assembly. Here, we investigate representative, acquired U2AF2 mutations, namely N196K or G301D amino acid substitutions associated with leukemia or solid tumors, respectively. We determined crystal structures of the wild-type (WT) compared with N196K- or G301D-substituted U2AF2 proteins, each bound to a prototypical AdML polypyrimidine tract, at 1.5, 1.4, or 1.7 Å resolutions. The N196K residue appears to stabilize the open conformation of U2AF2 with an inter-RNA recognition motif hydrogen bond, in agreement with an increased apparent RNA-binding affinity of the N196K-substituted protein. The G301D residue remains in a similar position as the WT residue, where unfavorable proximity to the RNA phosphodiester could explain the decreased RNA-binding affinity of the G301D-substituted protein. We found that expression of the G301D-substituted U2AF2 protein reduces splicing of a minigene transcript carrying prototypical splice sites. We further show that expression of either N196K- or G301D-substituted U2AF2 can subtly alter splicing of representative endogenous transcripts, despite the presence of endogenous, WT U2AF2 such as would be present in cancer cells. Altogether, our results demonstrate that acquired U2AF2 mutations such as N196K and G301D are capable of dysregulating gene expression for neoplastic transformation.
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Project description:The mammalian prions replicate by converting cellular prion protein (PrP(C)) into pathogenic conformational isoform (PrP(Sc)). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP(Sc) on conversion of PrP(C) in vitro using PrP(Sc) seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP(Sc). The smallest most potent prions present in sCJD brains were composed only of?20 monomers of PrP(Sc). The tight correlation between conversion potency of small oligomers of human sPrP(Sc) observed in vitro and duration of the disease suggests that sPrP(Sc) conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.
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Project description:RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III-related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development.
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Project description:The RNA exosome is a ubiquitously expressed complex of nine core proteins (EXOSC1-9) and associated nucleases responsible for RNA processing and degradation. Mutations in EXOSC3, EXOSC8, EXOSC9, and the exosome cofactor RBM7 cause pontocerebellar hypoplasia and motor neuronopathy. We investigated the consequences of exosome mutations on RNA metabolism and cellular survival in zebrafish and human cell models. We observed that levels of mRNAs encoding p53 and ribosome biogenesis factors are increased in zebrafish lines with homozygous mutations of exosc8 or exosc9, respectively. Consistent with higher p53 levels, mutant zebrafish have a reduced head size, smaller brain, and cerebellum caused by an increased number of apoptotic cells during development. Down-regulation of EXOSC8 and EXOSC9 in human cells leads to p53 protein stabilisation and G2/M cell cycle arrest. Increased p53 transcript levels were also observed in muscle samples from patients with EXOSC9 mutations. Our work provides explanation for the pathogenesis of exosome-related disorders and highlights the link between exosome function, ribosome biogenesis, and p53-dependent signalling. We suggest that exosome-related disorders could be classified as ribosomopathies.
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Project description:Prion diseases are caused by accumulation of an abnormally folded isoform (PrP(Sc)) of the cellular prion protein (PrP(C)). The subcellular distribution of PrP(Sc) and the site of its formation in brain are still unclear. We performed quantitative cryo-immunogold electron microscopy on hippocampal sections from mice infected with the Rocky Mountain Laboratory strain of prions. Two antibodies were used: R2, which recognizes both PrP(C) and PrP(Sc); and F4-31, which only detects PrP(C) in undenatured sections. At a late subclinical stage of prion infection, both PrP(C) and PrP(Sc) were detected principally on neuronal plasma membranes and on vesicles resembling early endocytic or recycling vesicles in the neuropil. The R2 labeling was approximately six times higher in the infected than the uninfected hippocampus and gold clusters were only evident in infected tissue. The biggest increase in labeling density (24-fold) was found on the early/recycling endosome-like vesicles of small-diameter neurites, suggesting these as possible sites of conversion. Trypsin digestion of infected hippocampal sections resulted in a reduction in R2 labeling of >85%, which suggests that a high proportion of PrP(Sc) may be oligomeric, protease-sensitive PrP(Sc).
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Project description:Neurogenic transcription factors and evolutionarily conserved signalling pathways have been found to be instrumental in the formation of neurons. However, the instructive role of microRNAs (miRNAs) in neurogenesis remains unexplored. We recently discovered that miR-9* and miR-124 instruct compositional changes of SWI/SNF-like BAF chromatin-remodelling complexes, a process important for neuronal differentiation and function. Nearing mitotic exit of neural progenitors, miR-9* and miR-124 repress the BAF53a subunit of the neural-progenitor (np)BAF chromatin-remodelling complex. After mitotic exit, BAF53a is replaced by BAF53b, and BAF45a by BAF45b and BAF45c, which are then incorporated into neuron-specific (n)BAF complexes essential for post-mitotic functions. Because miR-9/9* and miR-124 also control multiple genes regulating neuronal differentiation and function, we proposed that these miRNAs might contribute to neuronal fates. Here we show that expression of miR-9/9* and miR-124 (miR-9/9*-124) in human fibroblasts induces their conversion into neurons, a process facilitated by NEUROD2. Further addition of neurogenic transcription factors ASCL1 and MYT1L enhances the rate of conversion and the maturation of the converted neurons, whereas expression of these transcription factors alone without miR-9/9*-124 was ineffective. These studies indicate that the genetic circuitry involving miR-9/9*-124 can have an instructive role in neural fate determination.
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Project description:Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.
| S-EPMC3879332 | biostudies-literature