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Analysis of Fetal liver cells from hnRNP L KO mice

ABSTRACT: The regulation of post-transcriptional modifications of pre-mRNA by alternative splicing is important for cellular function, development and immunity. To investigate the role of hnRNP L in this process, we have generated conditional hnRNP L knockout mice carrying floxed alleles in order to generate a specific defect in hnRNP L activity. We have previously characterized defects present in the thymocytes of hnRNP L -/- mice and the current study extends the analysis to examine the effects in fetal liver cells. Overall these cells exhibit a very different phenotype from thymocytes characterized by significant cell death and a potential block in proliferation. Further functional studies were performed to better characterize these effects. RNA-seq from fetal liver cells of WT mice compared to KO (hnRNP L) mice

ORGANISM(S): Mus musculus

SUBMITTER: Brian Wilhelm

PROVIDER: E-GEOD-33815 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Alternative splicing controlled by heterogeneous nuclear ribonucleoprotein L (hnRNP L) regulates development, proliferation and migration of thymic pre-T cells

Project description:The regulation of post-transcriptional modifications of pre-mRNA by alternative splicing is important for cellular function, development and immunity The receptor tyrosine phosphatase CD45, which is expressed on all hematopoietic cells, is known for its role in the development and activation of T cells. To investigate the role of hnRNP L further, we have generated conditional hnRNP L knockout mice carrying floxed alleles and the T-cell specific Lck-Cre recombinase transgene. The Lck-Cre transgene is active in DN3, DN4 as well as in DP and SP cells. We found that deletion of hnRNP L results in a decreased thymic cellularity caused by a partial block at the transition stage between DN4 and DP cells. In addition, hnRNP L-/- thymocytes express aberrant levels of the CD45RA splice isoform and show high levels of phosphorylated Lck at the activator tyrosine Y394 but lacking phosphorylation of the inhibitory tyrosine Y505. This is indicative of an increased basal Lck activation and correlated with a higher proliferation rate of DN4 cells in hnRNP L-/- mice. Deletion of hnRNP L also blocked egress of SP cells to peripheral lymphoid organs and the migration of SP thymocytes in response to the chemokines CCL21 and SDF-1a. Since we found that actin polymerization was also compromised in hnRNP L-/- SP cells, we propose that a defect in the signal transduction cascade downstream of the chemokine receptors CCR7 and CXCR4 caused by the absence of hnRNP L is responsible for this phenotype. Our results indicate that hnRNPL regulates pre-T cell development and migration by regulating CD45 pre-mRNA splicing and chemokine receptor signaling. RNA-seq from Thymocytes from WT mice compared to KO (hnRNP L) mice

2012-04-23 | E-GEOD-33306 | biostudies-arrayexpress

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Analysis of Fetal liver cells from hnRNP L KO mice

Project description:The regulation of post-transcriptional modifications of pre-mRNA by alternative splicing is important for cellular function, development and immunity. To investigate the role of hnRNP L in this process, we have generated conditional hnRNP L knockout mice carrying floxed alleles in order to generate a specific defect in hnRNP L activity. We have previously characterized defects present in the thymocytes of hnRNP L -/- mice and the current study extends the analysis to examine the effects in fetal liver cells. Overall these cells exhibit a very different phenotype from thymocytes characterized by significant cell death and a potential block in proliferation. Further functional studies were performed to better characterize these effects.

2013-12-31 | GSE33815 | GEO

Alternative splicing controlled by heterogeneous nuclear ribonucleoprotein L (hnRNP L) regulates development, proliferation and migration of thymic pre-T cells

2012-04-24 | GSE33306 | GEO

HITS-CLIP analysis of FUS in lineage-committed cells.

Project description:In the present study, we performed HITS-CLIP analysis for FUS using mouse brain to extensively characterize tits RNA-binding sites and functional roles in RNA metabolisms. We identified preferential binding of FUS to stem-and-loop structures but without any discernible consensus motifs. FUS was preferentially bound to introns and 3' untranslated regions, but the exon/intron boundaries were mostly devoid of FUS-tags. Analysis of position-dependence of FUS-binding sites in regulating inclusion and skipping of exons disclosed that FUS is bound broadly around the alternatively spliced exons. Among them, however, noticeable CLIP-tags were observed in the downstream introns. We also noticed that FUS occasionally binds to the antisense strands in the promoter regions. Global analysis of CLIP-tags and expression profiles revealed that binding of FUS to the promoter antisense regions downgregulates transcription of the sense strand. HITS-CLIP (High Throughput Sequencing after Crosslinking and Immunoprecipitation) experiments targeting FUS in mouse cerebrums derived from 12-week-old C57BL/6 mice

2012-07-26 | E-GEOD-37190 | biostudies-arrayexpress

Composition of the mitoribosome of Diplonema papillatum

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Project description:Background: hnRNP K is an RNA-binding protein that's been implicated in oncogenesis, particularly in hematological disorders. Intent: To determine the impact of hnRNP K overexpression on RNA expression in murine fetal liver cells. Experimental Workflow: Fetal liver cells were subjected to retrovial transduction with either empty vector GFP or hnRNP K GFP. Purified GFP+ cells were flow sorted, and RNA was extracted from the GFP+ population, upon which RNA sequencing was performed.

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RNA-seq analysis of human heart failure

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Widespread N6-methyladenosine-dependent RNA Structural Switches Regulate RNA-Protein Interactions

Project description:We show that N6-methyladenosine (m6A), the most abundant internal modification in mRNA/lncRNA with still poorly characterized function, alters RNA structure to facilitate the access of RBM for heterogeneous nuclear ribonucleoprotein C (hnRNP C). We term this mechanism m6A-switch. Through combining PAR-CLIP with Me-RIP, we identify 39,060 m6A-switches among hnRNP C binding sites transcriptome-wide. We show that m6A-methyltransferases METTL3 or METTL14 knockdown decreases hnRNP C binding at 16,582 m6A-switches. Taken together, 2,798 m6A-switches of high confidence are identified to mediate RNA-hnRNP C interactions and affect diverse biological processes including cell cycle regulation. These findings reveal the biological importance of m6A and provide insights into the sophisticated regulation of RNA-RBP interactions through m6A-induced RNA structural remodeling. Measure the m6A methylated hnRNP C binding sites transcriptome-wide by PARCLIP-MeRIP; measure the differential hnRNP C occupancies upon METTL3/METTL14 knockdown by PAR-CLIP; measure RNA abundance and splicing level changes upon HNRNPC, METTL3 and METTL14 knockdown

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