Project description:To obtain a wholistic view of hnRNP-A2B1’s role in mRNA transport in response to DNA virus infection, we profiled RNAs from nuclear and cytoplasmic fractions in both wild-type and Hnrnpa2b1–/– macrophages after Herpes simplex virus 1 (HSV-1) infection.
Project description:Abstract Vault RNAs (vtRNAs) are a family of four small non-coding RNAs (ncRNAs) that are ubiquitously expressed in many eukaryotes and that regulate multiple cellular pathways. Their expression is increased upon infection with various DNA and RNA viruses. This suggests they either function to aid viral replication or as an antiviral restriction factor. However, their precise molecular function remains unclear. Here, we show that replication of picornaviruses, alphaviruses, and betacoronaviruses broadly enhances vtRNA expression. We find that genetic loss of vtRNAs inhibits replication of Sindbis virus (SINV) and encephalomyocarditis virus (EMCV) replication, independent of the antiviral type I interferon (IFN) response. A proteomic screen uncovered the vtRNA interactome and revealed that vtRNAs associate with RNA binding proteins ELAVL1 and hnRNP C in uninfected and infected cells. VtRNAs facilitate the translocation of ELAVL1 and hnRNP C from the nucleus to the cytoplasm in infected cells, an event that is required for efficient viral replication. Moreover, in the absence of vtRNAs, hnRNP C and ELAVL1 fail to associate with viral RNA in the cytosol of SINV-infected cells. Together, our findings uncover a novel molecular mechanism by which vtRNAs exert pro-viral activity during the course of SINV and EMCV infection, which opens up new avenues for therapeutic targeting to fight infectious diseases.
Project description:Identification of RNAs differentially expressed upon treatment with nontargeting siRNA, hnRNP L siRNA, UPF1 siRNA, or hnRNP L and UPF1 siRNAs together.
Project description:Macrophage activation by bacterial lipopolysaccharides (LPS) is induced through Toll-like receptor 4 (TLR4). The synthesis and activity of TLR4 downstream signalling molecules modulates the expression of pro- and anti-inflammatory cytokines. To address the impact of post-transcriptional regulation on that process, we performed RIP-Chip analysis. Differential association of mRNAs with heterogeneous ribonucleoprotein K (hnRNP K), an mRNA-specific translational regulator in differentiating haematopoietic cells, was studied in non-induced and LPS-activated macrophages. Analysis of interactions affected by LPS revealed an enrichment of mRNAs encoding TLR4 downstream kinases and their modulators. We focused on transforming growth factor β activated kinase-1 (TAK1), a central player in TLR4 signalling. HnRNP K interacts specifically with a sequence in the TAK1 mRNA 3' UTR in vitro. Silencing of hnRNP K does not affect TAK1 mRNA synthesis and stability, but enhances TAK1 mRNA translation, resulting in elevated TNF-alpha, IL-1beta and IL-10 mRNA expression. Our data suggest that the hnRNP K-3' UTR complex inhibits TAK1 mRNA translation in non-induced macrophages. LPS-dependent TLR4 activation abrogates translational repression and newly synthesised TAK1 initiates the inflammatory response of macrophages. In this dataset, we include expression data of RAW 264.7 cells comparing untreated cells and 6h Lipopolysaccharide treatment, and analyse RNAs co-precipitating with hnRNP K dependent on LPS treatment. 12 total samples were analyzed, 6 samples of 2 biological replicates.
Project description:Heterogeneous nuclear ribonucleoproteins (hnRNPs) are involved in many processes in RNA metabolism. In addition to their functions in the nucleus, hnRNPs can function in the replication of RNA viruses in the cytoplasm. In vesicular stomatitis virus (VSV)-infected cells, several hnRNPs relocalize from the nucleus to the cytoplasm. This raises the question of whether these hnRNPs are relocalized together with their host nuclear RNAs or whether they associate with new RNAs in the cytoplasm. hnRNP A1, hnRNP C1/C2, and hnRNP K were immunoprecipitated from mock- or VSV-infected cells, and RNAs were analyzed by high content RNA sequencing. Each hnRNP displayed a loss of interaction with cellular transcripts in favor of viral mRNAs. hnRNP A1 was preferentially associated with VSV phosphoprotein (P) mRNA; hnRNP C1/C2 was preferentially associated with VSV glycoprotein (G) mRNA, and hnRNP K bound viral transcripts in rank of abundance.
Project description:Macrophage activation by bacterial lipopolysaccharides (LPS) is induced through Toll-like receptor 4 (TLR4). The synthesis and activity of TLR4 downstream signalling molecules modulates the expression of pro- and anti-inflammatory cytokines. To address the impact of post-transcriptional regulation on that process, we performed RIP-Chip analysis. Differential association of mRNAs with heterogeneous ribonucleoprotein K (hnRNP K), an mRNA-specific translational regulator in differentiating haematopoietic cells, was studied in non-induced and LPS-activated macrophages. Analysis of interactions affected by LPS revealed an enrichment of mRNAs encoding TLR4 downstream kinases and their modulators. We focused on transforming growth factor β activated kinase-1 (TAK1), a central player in TLR4 signalling. HnRNP K interacts specifically with a sequence in the TAK1 mRNA 3' UTR in vitro. Silencing of hnRNP K does not affect TAK1 mRNA synthesis and stability, but enhances TAK1 mRNA translation, resulting in elevated TNF-alpha, IL-1beta and IL-10 mRNA expression. Our data suggest that the hnRNP K-3' UTR complex inhibits TAK1 mRNA translation in non-induced macrophages. LPS-dependent TLR4 activation abrogates translational repression and newly synthesised TAK1 initiates the inflammatory response of macrophages. In this dataset, we include expression data of RAW 264.7 cells comparing untreated cells and 6h Lipopolysaccharide treatment, and analyse RNAs co-precipitating with hnRNP K dependent on LPS treatment.
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.
Project description:To inform the mechanism of hnRNP H dysfunction in methamphetamine-induced dopamine release and behavior, we surveyed mRNA targets of hnRNP H via cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in striatal tissue at baseline and at 30 min post-MA (2 mg/kg, i.p.). To integrate identification of hnRNP H targets with the impact of methamphetamine on downstream gene expression and splicing, we analyzed the transcriptome of the parallel samples used in CLIP-seq.