RESUMO
Germ cells contain non-membrane bound cytoplasmic organelles that help maintain germline integrity. In C. elegans they are called P granules; without them, the germline undergoes partial masculinization and aberrant differentiation. One key P-granule component is the Argonaute CSR-1, a small-RNA binding protein that antagonizes accumulation of sperm-specific transcripts in developing oocytes and fine-tunes expression of proteins critical to early embryogenesis. Loss of CSR-1 complex components results in a very specific, enlarged P-granule phenotype. In a forward screen to identify mutants with abnormal P granules, ten alleles were recovered with a csr-1 P-granule phenotype, eight of which contain mutations in known components of the CSR-1 complex (csr-1, ego-1, ekl-1, and drh-3). The remaining two alleles are in a novel gene now called elli-1 (enlarged germline granules). ELLI-1 is first expressed in primordial germ cells during mid-embryogenesis, and continues to be expressed in the adult germline. While ELLI-1 forms cytoplasmic aggregates, they occasionally dock, but do not co-localize with P granules. Instead, the majority of ELLI-1 aggregates accumulate in the shared germline cytoplasm. In elli-1 mutants, several genes that promote RNAi and P-granule accumulation are upregulated, and embryonic lethality, sterility, and RNAi resistance in a hypomorphic drh-3 allele is enhanced, suggesting that ELLI-1 functions with CSR-1 to modulate RNAi activity, P-granule accumulation, and post-transcriptional expression in the germline.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Grânulos Citoplasmáticos/metabolismo , Células Germinativas/metabolismo , Interferência de RNA , Fatores Genéricos de Transcrição/genética , Alelos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Mutação , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Genéricos de Transcrição/metabolismoRESUMO
Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Germinativas/fisiologia , Espermatozoides/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Masculino , Interferência de RNA/fisiologia , Espermatogênese/genética , Espermatogênese/fisiologiaRESUMO
ß2-adrenoreceptor (ß2AR) agonists have been associated with a decreased risk of developing Parkinson's disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of ß2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.
RESUMO
OpenArray is one of the most high-throughput qPCR platforms available but its efficiency can be limited by sample preparation methods that are slow and costly. To optimize the sample workflow for high-throughput qPCR processing by OpenArray, small-scale sample preparation methods were compared for compatibility with this system to build confidence in a method that maintains quality and accuracy while using less starting material and saving time and money. This study is the first to show that the Cells-to-CT kit can be used to prepare samples within the dynamic range of OpenArray directly from cultured cells in a single well of a 96-well plate when used together with a cDNA preamplification PCR step. Use of Cells-to-CT produced results of similar quality and accuracy to that of a preparation method using purified RNA in less than half the sample preparation time. While Cells-to-CT samples also exhibited slightly increased variance, which affects the ability of OpenArray to distinguish small differences in gene expression, overall gene expression mean results correlated well between small-scale methods. This work demonstrates that Cells-to-CT with preamplification can be used to reliably prepare samples for OpenArray analysis while saving time, money, and starting material.
RESUMO
In Caenorhabditis elegans, germline expression programs are actively repressed in somatic tissue by components of the synMuv (synthetic multi-vulva) B chromatin remodeling complex, which include homologs of tumor suppressors Retinoblastoma (Rb/LIN-35) and Malignant Brain Tumor (MBT/LIN-61). However, the full scope of pathways that suppress germline expression in the soma is unknown. To address this, we performed a mutagenesis and screened for somatic expression of GFP-tagged PGL-1, a core P-granule nucleating protein. Eight alleles were isolated from 4000 haploid genomes. Five of these alleles exhibit a synMuv phenotype, whereas the remaining three were identified as hypomorphic alleles of known synMuv B genes, lin-13 and dpl-1. These findings suggest that most suppressors of germline programs in the soma of C. elegans are either required for viability or function through synMuv B chromatin regulation.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alelos , Animais , Animais Geneticamente Modificados , Mapeamento Cromossômico , Expressão Gênica , Genes Reporter , Células Germinativas , Mutação , Interferência de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans [1]. In Drosophila, germ granules have been implicated in germline determination [2]. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination [3], but their components are still needed for fertility [4-6]. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 or upon neuronal induction by CHE-1 [7, 8]. However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, we severely compromised P granules by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization (GLH-1 and GLH-4) [9] and investigated whether this causes germ cells to lose totipotency and initiate somatic reprogramming. We found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate.