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HuR- dependent regulation of mRNA splicing is essential for the B cell antibody response [RNA-Seq]

ABSTRACT: Post-transcriptional regulation of mRNA by the RNA binding protein HuR is required in B cells for the germinal centre reaction and for the production of class-switched antibodies in response to T-independent antigens. Transcriptome-wide examination of RNA isoforms, abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interaction, revealed that HuR-dependent mRNA splicing affects hundreds of transcripts including the dihydrolipoyl succinyltransferase (Dlst), a subunit of the aketoglutaratedehydrogenase (aKGDH) enzyme. In the absence of HuR, defective mitochondrial metabolism results in high levels of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for B cell proliferation and differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE62129 | GEO | 2015/02/13

SECONDARY ACCESSION(S): PRJNA263260

REPOSITORIES: GEO

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HuR- dependent regulation of mRNA splicing is essential for the B cell antibody response [Ribo-Seq]

Project description:Post-transcriptional regulation of mRNA by the RNA binding protein HuR is required in B cells for the germinal centre reaction and for the production of class-switched antibodies in response to T-independent antigens. Transcriptome-wide examination of RNA isoforms, abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interaction, revealed that HuR-dependent mRNA splicing affects hundreds of transcripts including the dihydrolipoyl succinyltransferase (Dlst), a subunit of the aketoglutaratedehydrogenase (aKGDH) enzyme. In the absence of HuR, defective mitochondrial metabolism results in high levels of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for B cell proliferation and differentiation.

2015-02-13 | GSE62134 | GEO

HuR- dependent regulation of mRNA splicing is essential for the B cell antibody response [iCLIP-Seq]

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HuR- dependent regulation of mRNA splicing is essential for the B cell antibody response [RNA-Seq]

2015-02-13 | E-GEOD-62129 | biostudies-arrayexpress

HuR- dependent regulation of mRNA splicing is essential for the B cell antibody response [Ribo-Seq]

2015-02-13 | E-GEOD-62134 | biostudies-arrayexpress

Rip-Chip profiling of HnRNP A1 nuclear complexes in HuR WT and HuR KO cells

Project description:Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR (such as transport, transcription, translation, apoptosis and cell cycle) indicative of their concerted function in mRNA metabolism. Ribonucleoprotein Immunoprecipitation (RIP) using hnRNP A1 specific antibody was performed in nuclear extracts from HuR WT and HuR KO Mouse Embryonic Fibroblasts (MEFs). RNA isolated from these IPs together with nuclear RNA from the two cell types, was subjected to microarray analysis. Three biological replicates, representing three independent experiments, are available for each condition except in the case of nuclear RNA isolated from HuR WT MEFs that one replicate didn’t pass the quality control.

2013-11-01 | E-GEOD-49225 | biostudies-arrayexpress

Rip-Chip profiling of HnRNP A1 nuclear complexes in HuR WT and HuR KO cells

2013-11-01 | GSE49225 | GEO

The RNA-binding protein HuR is required for antigen-specific germinal centre B cell selection.

Project description:The germinal centre (GC) is a hallmark of adaptive immunity. Gene expression in GCs is tightly regulated but the impact of post-transcriptional mechanisms are largely unknown. Here we show that expression of the RNA binding protein HuR is essential in GCs. In its absence, GC B cells are at a competitive disadvantage and high-affinity antibody production is severely impaired. Mechanistically, HuR affects the transcriptome qualitatively and quantitatively to enable the expression of a Myc- dependent transcriptional program essential for light zone B cells to re-enter into the dark zone for further proliferation and Ig somatic hypermutation. HuR controls the splicing and abundance of mRNAs required for entry into and transition through the S phase of the cell cycle. HuR modulates a gene signature associated with DNA deamination preserving GC B cells from extensive DNA damage and cell death.

2021-09-22 | GSE145413 | GEO

RIP-chip of HuR in MCF7 cells exposed to doxorubicin

Project description:The purpose of the study was to identify mRNA bound to HuR in the presence of doxorubicin in MCF7 cells. We collected cytoplasmic RNA from untreated and treated cells and detected differentially expressed genes (DEGs). We also coimmunoprecipitated HuR and IgG (as control) from doxorubicin treated cells. Comparison between HuR RIP and IgG RIP signals was used to discriminate specific mRNA bound to HuR. HuR coimmmunoprecipitated material was hybridized together with cytoplasmic mRNA of doxorubicin treated cells, enabling the fold enrichment calculation and the selection of mRNAs bound to HuR. Keywords: RIP-Chip, HuR, doxorubicin, MCF7, HuR consensus binding, post-transcriptional regulation.

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Unstressed HeLa cells and ELAVL1/HuR knock down conditions: polyA RNA-Seq, small RNA-Seq, and PAR-CLIP

Project description:Post-transcriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a method based on RNA-protein crosslinking, to identify transcriptome wide ~26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in splicing. Upon HuR knock down, mRNA levels and protein synthesis of thousands of target genes was down regulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knock down triggered strong and specific up regulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing. PolyA mRNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression and splicing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq. PARCLIP was performed as in Hafner et. Al 2010 but with an antibody against endogenous HuR (3A2, Santa Cruz, sc-5261) in unstressed HeLa cells. We used, independently, 4-thiouridine (4SU) and 6-thioguanosine (6SG) to assess a possible nucleotide bias. As our proteomics measurements required labeling of cells in a special medium we also performed PAR-CLIP on cells grown in SILAC medium. Altogether we performed three PAR-CLIP experiments: 4SU labeling in standard DMEM medium, 4SU labeling in SILAC medium (as a replicate) and 6SG labeling in SILAC medium. Small RNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression. Sequencing was performed on Illumina GAII using the standard sRNA 36cycle protocol.

2011-06-29 | E-GEOD-29943 | biostudies-arrayexpress

HuR (ELAVL1) RIP-chip and knockdown exon array

Project description:Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability In this dataset, we employed two distinct experiments. 1) HuR RIP-chip to identify mRNA targets of HuR. 2) HuR knockdown to identify mRNAs whose expression are dependent on HuR.

2011-08-19 | GSE29778 | GEO

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