Project description:High-throughput sequencing analysis of transcriptome from LPS-activated B cells from Lymph node of Zfp36l1(fl/fl) and Zfp36l1(fl/fl) x Cd79a(Cre/+) mice
Project description:We have analyzed miRNA expression profile in FO and MZ B cells to identified differentially expressed miRNAs between this two subsets that could be potencially involved in the regulation of terminal B cell differentiation Spleens from CD19-Creki/+Dicerfl/+ and CD19-Creki/+Dicerfl/fl mice were collected and after erythrocyte lysis, splenocytes were stained with anti-B220, anti-CD21 and anti-CD23 antibodies. B220+CD21brightCD23+ (MZ) and B220+CD21+CD23bright (FO) B cell populations were sorted and total RNA of purified populations was extracted with TRIzol. miRNA microarray hybridations were performed on Mouse miRNA Microarray platform (Agilent Technologies).
Project description:We have analyzed miRNA expression profile in FO and MZ B cells to identified differentially expressed miRNAs between this two subsets that could be potencially involved in the regulation of terminal B cell differentiation
Project description:Purpose: We aimed to identify the targets of the RNA binding protein ZFP36L1 in thymoctes. Methods: Total naïve thymocytes from control or DCKO mice were treated with UV light to crosslink RNA and proteins, then RNA-protein complexes were pulled down with anti-ZFP36L1. RNA was extracted and used to make cDNS libraries that were then sequenced by MiSeq 150bp single-end read (Sample 1) and HiSeq2500 RapidRun 50bp single-end read (sample 2, 3). Results: Sample demultiplexing was performed by identification of the 3 known bases of the 7 bases barcode introduced in the 5â end of the read by the RCLIP primer. The remaining four random bases were used to remove PCR duplicate reads. Reads were trimmed to remove any adaptor sequence and barcodes before mapping reads to genome GRCm38 using Bowtie. After read mapping, the single-nucleotide at position -1 was annotated as unique ZFP36L1 crosslink site. Identification of highly significant ZFP36L1 binding sites was performed using iCount to assign a FDR to each crosslink site. Conclusions: We identified ZFP36L1 binding sites in 8675 thymocyte mRNAs. Individual nucleotide resolution cross linking immunoprecipitation (iCLIP) of ZFP36L1-bound RNAs in total naive thymocytes from C57BL/6 mice.
Project description:Purpose: The goal of this study was to assess if increased TLR7 signaling could be seen in TLR9-/- compared to TLR9WT FO and MZ B cells. Methods : FO and MZ B cells from TLR9WT or TLR9-/- BALB/c mice were sorted using FACSaria. A minimum of 100,000 cells per B cell subsets were sorted. Sorted B cell subsets were stimulated for 4 hours at 0.25M/ml with TLR7 agonist CL097 (Invivogen) at 5µg/ml or left unstimulated. RNA was isolated using the RNeasy Plus Micro Kit (QIAGEN). Samples were sequenced on an Illumina Next-seq 2000 using a P3 200 cycle flowcell (Illumina, Inc) with 2 x 101 bp paired-end reads (20 million reads per sample). Results: There were no significant DEG (FDR > 0.05) following TLR7 stimulation between TLR9WT and TLR9-/- FO or MZ B cells.
Project description:ZFP36L1 is a tandem zinc-finger RNA-binding protein that recognizes conserved Adenylate-Uridylate-rich Elements (AREs) located in 3' untranslated regions (UTRs) to mediate RNA decay. We hypothesized that ZFP36L1 is a negative regulator of a post-transcriptional hub involved in the RNA half-life regulation of cancer-related transcripts. Forced expression of ZFP36L1 in cancer cells markedly reduced cell proliferation in vitro and in vivo; whereas silencing of ZFP36L1 enhanced tumor cell growth. To identify direct downstream targets of ZFP36L1, systematic screening using RNA pull-down of wildtype and mutant ZFP36L1 as well as whole transcriptome sequencing of bladder cancer cells ± tet-on ZFP36L1, was performed. A network of 1,410 genes was identified as potential direct targets of ZFP36L1, including HIF1A, CCND1, and E2F1. ZFP36L1 specifically bound to the 3' UTRs of these targets for RNA degradation, thus suppressing their expression. Collectively, our findings reveal an indispensable role of ZFP36L1 as a post-transcriptional safeguard against aberrant hypoxic signaling and abnormal cell cycle progression.