RNA seq of microglia from CPEB1-deficient and LPS-treated mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE195513 | GEO | 2022/08/24
REPOSITORIES: GEO
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Project description:RNA seq of microglia from CPEB1-deficient and LPS-treated mice
| PRJNA800903 | ENA
Project description:CPEB1 regulates cellular function by post-transcriptionally controlling its targeted transcripts' translation. After bound to CPEs, CPEB1 recruits cytoplasmic poly (A) polymerase GLD2 to elongate the poly (A) tail for the maintenance of mRNA stability. The stability of mRNA is positively correlated with translational output. It is unexamined whether CPEB1 interacts with translation machinery to regulation translation. Previous reports demonstrate that phosphorylation is required for its regulation on translation. However, it is not well understood whether phosphorylation is essential for CPEB1 interaction with translation machinery or not. Here, we performed mass spectrometry on C2 cells transfected with CPEB1-mVenus or CPEB1 (T171A, S177A)-mVenus after IgG or mVenus antibody immunoprecipitation. After analysis, we identify CPEB1 interacting proteins and the phosphorylation dependent interacting proteins such as ribosomal proteins. Thus, our data suggest that CPEB1 regulate translation by interacting with translation machinery in a phosphorylation dependent manner.
2021-11-04 | PXD020822 | Pride
Project description:Poly(A) tail length is regulated in both the nucleus and cytoplasm. One factor that controls polyadenylation in the cytoplasm is CPEB1, an RNA binding protein that associates with specific mRNA 3’UTR sequences to tether enzymes that add and remove poly(A). Two of these enzymes, the noncanonical poly(A) polymerases Gld2 (TENT2, PAPD4, Wispy) and Gld4 (TENT4B, PAPD5, TRF4, TUT3), interact with CPEB1 to extend poly(A). To identify additional RNA binding proteins that might anchor Gld4 to RNA, we expressed double tagged Gld4 in U87MG cells, which was used for sequential immunoprecipitation and elution followed by mass spectrometry. We identified several RNA binding proteins that co-precipitated with Gld4, among which was FMRP. To assess whether FMRP regulates polyadenylation, we performed TAIL-seq from WT and FMRP-deficient HEK293 cells. Surprisingly, loss of FMRP resulted in an overall increase in poly(A), which was also observed for several specific mRNAs. Conversely, loss of CPEB1 elicited an expected decrease in poly(A), which was examined in cultured neurons. We also examined polyadenylation in wild type (WT) and FMRP-deficient mouse brain cortex by direct RNA Nanopore™ sequencing, which identified RNAs with both increased and decreased poly(A). Our data show that FMRP has a role in mediating poly(A) tail length, which adds to its repertoire of RNA regulation.
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Project description:Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes, many of which have been implicated in several diseases and disorders of the central nervous system including alcohol use disorders and disorders. By utilizing a method optimized for sensitive and rapid quantitative proteomic analysis of microglia involving suspension trapping (S-Trap), we were able to produce efficient and reproducible protein extraction from low cell yielding primary mouse brains. Using a ~2 h gradient on a 75 cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer (QE Plus), 5,062 total proteins were identified where 4,928 of those proteins were quantifiable by label-free quantitation (with 5 biological replicates). This analysis resulted in the most comprehensive proteomic dataset for ethanol- and LPS-treated primary mouse microglia to date and even expanded upon the well-characterized macrophage/microglia response to LPS treatment. This study also highlights the subtle, yet significant changes ethanol exposure can induce when compared to control. Interestingly, these changes are not consistent with the robust classical activation induced by LPS treatment, but instead align with the emerging theory that ethanol-treated microglia yield an alternative activation response. The contrast to LPS-treated microglia leads us to conclude that ethanol does not elicit a strong inflammatory response but rather might have a general inhibitory effect on multiple pathways such as phagocytosis and cell migration.
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Project description:Primary microglia were derived from neonatal mice and were activated via LPS+ATP treatment. test mice were treated with the drug Ladostigil and transcriptome was compared to untreated controls
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Project description:The experiment is to demonstrate the global m6A methylation signatures and their correlation with mRNA expression patterns in microglia stimulated by LPS. The results also identified m6A modifiers with significantly altered expression, which reveals the key one that regulate the m6A modifications in microglia.
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Project description:We compared the poly(A) tail length status of mRNAs of HeLa cells expressing a CPEB1 shRNA (CPEB1 knockdown) versus a control shRNA, and expressing a CPEB4 shRNA (CPEB4 knockdown) versus a control shRNA. Results provide insight into the extent of gene regulation mediated by CPEB1 and CPEB4 activity during mitotic cell cycle progression. The different shRNA expressing cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and samples were taken after 8 hours release (G2/M phase). For each shRNA expressing HeLa cell line total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 μg total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261. Keywords: knock-down experiment
2010-04-01 | GSE15549 | GEO
Project description:We investigated the expression patterns of transcripts in mouse primary microglia that are stimulated by LPS and transfected with miR-106b mimics. Three experiment groups were utilized in the RNA-seq, that are PBS+mimics control group, LPS+mimics group, and LPS+miR-106b mimics group. Samples were collected 2 days after LPS treatment and mimics transfection.
2021-10-31 | E-MTAB-10946 | biostudies-arrayexpress
Project description:Ischemic stroke is a common acute CNS disorder leading to nearly half a million deaths per year in Europe. The high mortality is primarily owed to the limited treatment options of restoring blood flow in a narrow time window of several hours. Furthermore, inflammatory processes in the days and weeks after ischemic stroke contribute to tissue loss and neurological deficits. The key cells that influence and control this inflammatory cascade are microglia, the innate immune cells of the CNS. Microglia can be influenced and activated by e.g. lipopolysaccharide (LPS),a bacterial cell membrane component. It has been previously shown, that repetitive LPS stimuli prior to infarction (termed immunological preconditioning) lead to reduced infarct volumina in mouse models of ischemic stroke. Furthermore, our laboratory has shown, that phosphoinositide-3 kinase gamma mediates microglial functions after LPS-preconditioning. Hence, the aim of this work was to characterize proteomic alterations in microglia with (I) ischemic stroke in general in the tMCAO (transient middle cerebral artery occlusion) mouse model of ischemic stroke, (II) the influence of LPS-preconditioning on microglial proteomic alterations after tMCAO and (III) the role of PI3Ky in the microglial proteomic changes after tMCAO and preconditioning. This was done by a single LPS injection 3 days before tMCAO in wildtype mice and mice with PI3Ky knockout or knockin of the kinase dead form of PI3Ky.
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Project description:Several independent microglia preparations performed on different days were plated onto wells of 24-well plates, and either left untreated (control), treated with the C6 glioma-conditioned medium, or treated with LPS.<br>For each culture condition, a number of RNA samples were prepared, with each sample then separately labeled and hybridized. These RNA samples were prepared from non-overlapping sets of wells from at least two microglia preparations, so they constitute biological replicates.
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