Project description:To explore the effect of LIN28 (S200) phosphorylation on LIN28's association with mRNAs on a transcriptome-wide scale, we performed mRNA-seq on RNAs immunoprecipitated (RIP-seq) by wild-type (WT) or phospho-null (S200A) LIN28 in HeLa cells.
Project description:To explore the effect of LIN28 (S200) phosphorylation on LIN28's association with mRNAs on a transcriptome-wide scale, we performed mRNA-seq on RNAs immunoprecipitated (RIP-seq) by wild-type (WT) or phospho-mimetic (S200D) LIN28 in PA1 cells.
Project description:The RNA-binding protein LIN28A is required for maintaining tissue homeostasis, including in the reproductive system, but the underlying mechanisms on how LIN28A regulates germline progenitors remain unclear. Here, we dissected LIN28A-binding targets using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse testes. LIN28A preferentially binds to mRNA coding sequence (CDS) or 3'UTR regions at sites enriched wiGAG(A) sequences. Further investigation of Lin28a null mouse testes indicated that meiosis-associated mRNAs bound by LIN28A were differentially expressed. Next, ribosome profiling revealed that the mRNA levels of these targets were significantly reduced in polysome fractions, and their protein expression levels decreased in the Lin28a null mouse testes, even when meiotic arrest in the null mouse testes was not apparent. Collectively, these findings provide a set of LIN28A-regulated target mRNAs, and show that LIN28A binding might be mechanism through which LIN28A acts to regulate undifferentiated spermatogonia fates and male fertility in mammals.
Project description:WT and TorsinKO HeLa cells were transfected with d133-ORF10-FLAG from KSHV. The protein was immunoprecipitated with anti-FLAG beads to investigate proteins contained within nuclear envelope blebs. A control sample with no specific immunoprecipitation was compared to the co-immunoprecipitations. Eluting proteins were ran into an SDS-PAGE gel and extracted for mass spectrometry analysis.
Project description:Purpose: The goals of this study are to investigate the mRNAs that bind to the FTO protein in the white fat tissue from mice. Methods: 1. White fat from CAG promoter driven transgenic Flag-tagged FTO mice were extracted with RNA protected. 2. Flag-FTO proteins were immunoprecipitated with control IP using IgG. 3. The immunoprecipitated RNAs were deep sequenced and analyzed. Results: We focused on the mRNAs which have functions related to lipid metabolism and homeostasis.
Project description:To determine if changes in Protein Disulfide Isomerase (PDIA1) expression in mice with a null Reelin allele were caused by accumulation of intracellular Reelin or were an effect of reduced Reelin protein, we examined expression of PDIA1 and other stress markers in heterozygous RELN +/- null allele mice. The levels of PDIA1 as well as PERK, BIP, phospho-eIF2alpha and total eIF2alpha were unchanged between wild-type and RELN +/- null allele mice. This suggested that there are phenotypic differences in the cerebella between mice that carry a RELN allele that fails to produce a protein (null allele) and those that make a protein that fails to be secreted (Orl allele). Each of the three major ER stress pathways ultimately leads to changes in gene transcription. Thus, we compared wild-type and heterozygous RELN Orl +/- mice cerebellum by RNAseq. Analysis was performed on 3 heterozygous (HET) and 3 wild-type (WT) cerebella, obtained from 6-week old male mice.
Project description:Proximity labelling using transient expression of the same exogenous BirA*-RAB18 construct in wild-type HeLa cells and in otherwise isogenic RAB3GAP1-, RAB3GAP2- and TRAPPC9-null HeLa cell lines.
Project description:Isolation of IMP1 bound mRNAs. Flag-tagged IMP1 was expressed in HEK293 cells. Flag tagged IMP1 was immunoprecipitated and mRNAs isolated. As controls HEK293 cells that do not express Flag-tagged IMP1 was included.
