Project description:Higher N(6)-methyladenosine (m6A) was identified in selected primary microRNAs (pri-miRNAs) as associated with increased levels of the corresponding mature miRNA in MDA-MB-231 triple negative breast cancer (TNBC) cells (PMID: 25799998). HNRNPA2B1 is a reader of the m6A mark in pri-miRNAs and interacts directly with DGCR8 (a component of the nuclear DROSHA complex) to promote processing of pri-miRNAs to precursor-miRNAs (pre-miRNAs) (PMID: 26321680). We have preliminary data showing that HNRNPA2B1 is increased in TAM-resistant, ERα+ LCC9 cells derived from TAM-sensitive, ERα+ MCF-7 breast cancer cells. We found that high HNRNPA2B1 transcript levels in primary breast tumors are associated with reduced overall survival. We postulate that an increase in HNRNPA2B1 targets its binding to additional pri-miRNAs (not normally bound under wild type conditions), resulting in an increase in miRNAs, including those that promote TAM-resistance.
Project description:To identify HNRNPA2B1 binding sites on endogenous nuclear RNAs, we performed HITS-CLIP for endogenous HNRNPA2B1 and RNA-seq to analyze the nuclear RNA under either METTL3 or HNRNPA2B1 depletion. Wild type MDA-MB-231 cells were subjected to the HITS-CLIP procedure on immunoprecipitated HNRNPA2B1 associated RNA obtained from the nuclear fraction (Licatalosi D, et al. 2008, Nature 456:464-U22). For RNA-seq, nuclear RNA was extracted from MDA-MB-231 or Hela cells knocked down for METTL3 or HNRNPA2B1.
Project description:Exosomes are released by most cells to the extracellular environment, and are involved in cell-to-cell-communication. Exosomes contain specific repertoires of mRNAs, miRNAs and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe short sequence motifs present in miRNAs that control their localization into exosomes. The protein hnRNPA2B1 specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications.
Project description:To identify HNRNPA2B1 binding sites on endogenous nuclear RNAs, we performed HITS-CLIP for endogenous HNRNPA2B1 and RNA-seq to analyze the nuclear RNA under either METTL3 or HNRNPA2B1 depletion.
Project description:miRNAs regulate mRNA stability and translation through the action of the RNAi-induced silencing complex. In this study, we systematically identified endogenous miRNA target genes by using AGO2 immunoprecipitation (AGO2-IP) and microarray analyses in two breast cancer cell lines, MCF7 and MDA-MB-231, representing luminal and basal-like breast cancer, respectively. The expression levels of ~70% of the AGO2-IP mRNAs were increased by DROSHA or DICER1 knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3'-UTR of mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the fifty most abundantly expressed miRNAs.
Project description:miRNAs are short regulatory single stranded RNA sequences that upon complementary binding to mRNAs lead to the inhibition or degradation of their targets. This regulatory mechanisms has been shown to play crucial roles throughout the whole life cycle of animals and plants as well as in disease. While a plethora of methods exist to predict targets of miRNA, which suggest that up to 80% of the genome is miRNA regulated, it has recently been reported that many of these predictions are false positives, cell type specific or represent non-functional binding. In order to identify the subset of real functional miRNAs and their targets, we established miRNA pathway mutants in mouse embryonic stem cells (mESC), allowing the dissection of canonical and non-canonical functions of pathway members. Additional data integration of downstream regulatory layers (CLIP-seq, ribosome profiling and MS) enabled us to follow and track down real functional miRNA-gene interactions, which reduced the miRNA genome regulation to approximately 1%.
Project description:miRNAs regulate mRNA stability and translation through the action of the RNAi-induced silencing complex. In this study, we systematically identified endogenous miRNA target genes by using AGO2 immunoprecipitation (AGO2-IP) and microarray analyses in two breast cancer cell lines, MCF7 and MDA-MB-231, representing luminal and basal-like breast cancer, respectively. The expression levels of ~70% of the AGO2-IP mRNAs were increased by DROSHA or DICER1 knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3'-UTR of mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the fifty most abundantly expressed miRNAs. To identify mRNAs associated with AGO2, cell lysate was precleaned with control IgG and immunoprecipitated with anti-human Ago2 (Clone 2E12-1C9, Abnova). Total RNA from cell lysate or coimmunoprecipitated with AGO2 was extracted with Trizol and subjected to microarray analysis, with three biological repeats for each experimental condition.
Project description:miRNAs regulate mRNA stability and translation through the action of the RNAi-induced silencing complex. In this study, we systematically identified endogenous miRNA target genes by using AGO2 immunoprecipitation (AGO2-IP) and microarray analyses in two breast cancer cell lines, MCF7 and MDA-MB-231, representing luminal and basal-like breast cancer, respectively. The expression levels of ~70% of the AGO2-IP mRNAs were increased by DROSHA or DICER1 knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3'-UTR of mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the fifty most abundantly expressed miRNAs.
Project description:To investigate the role of hnRNPA2B1 in the progression of hepatocellular carcinogenesis, we established a Huh7 cell line in which the hnRNPA2B1 gene was knocked out by crisper/cas9.