Project description:SYNCRIP, a member of the cellular heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA binding proteins, regulates various aspects of neuronal development and plasticity. Although SYNCRIP has been identified as a component of cytoplasmic RNA granules in dendrites of mammalian neurons, only little is known about the specific SYNCRIP target mRNAs that mediate its effect on neuronal morphogenesis and function. Here, we present a comprehensive characterization of the cytoplasmic SYNCRIP mRNA interactome using iCLIP in primary rat cortical neurons. We identify hundreds of bona fide SYNCRIP target mRNAs, many of which encode for proteins involved in neurogenesis, neuronal migration and neurite outgrowth. From our analysis, the stabilization of mRNAs encoding for components of the microtubule network, such as doublecortin (Dcx), emerges as a novel mechanism of SYNCRIP function in addition to the previously reported control of actin dynamics. Furthermore, we found that SYNCRIP interacts with miRISC and synergizes with pro-neural miRNAs, such as miR-9. Based on our findings, we propose a model whereby SYNCRIP promotes early neuronal differentiation by a two-tier mechanism involving the stabilization of pro-neural mRNAs by direct 3’UTR interaction and the repression of anti-neural mRNAs in a complex with neuronal miRISC. Together, our findings provide a rationale for future studies investigating the function of SYNCRIP in mammalian brain development and disease.
Project description:T cell activation leads to dramatic changes in cellular phenotype. We used CD3/CD28-activated human CD4 T cells to study how RNA binding proteins define the post-transcriptional landscape. Using RIPseq, we identified the RNA interactome of U2AF2 and show at the global level that U2AF2 binds the majority of transcripts that are differentially expressed and/or alternatively spliced during CD4 T cell activation. A unique protein interactome centered on U2AF2 is assembled in response to activation. Knocking down specific U2AF2 interacting partners (U2AF1, SYNCRIP, SRRM2, ILF2) selectively affects cytokine secretion and expression of activation markers. Furthermore, the expression and/or alternative splicing of transcripts important for immune cell function are also affected by knocking down these U2AF2 interacting proteins. U2AF1 and SYNCRIP knockdowns affect the proteins and transcripts bound to U2AF2, altering the transcriptome. Our work highlights the importance of RNA binding protein complexes in regulating the differential expression and alternative splicing that defines T cell activation. HTA 2.0 microarray results of U2AF2 RIP RNA from a primary CD4 T cell culture after treatment with siRNAs (Control, U2AF1, and SYNCRIP) and 24 hours after anti-CD3/CD28 bead activation Please note that the Series supplementary 'U2AF2RIP_HTA2_all_siRNA_100414.xlsx' file includes multiple worksheets containing: 1) Differentially expressed genes in ctrl siRNA vs. U2AF1 siRNA; 2) Differntially spliced genes in ctrl siRNA vs. U2AF1 siRNA; 3) Differentially spliced exons in ctrl siRNA vs. U2AF1 siRNA; 4) Differentially expressed genes in ctrl siRNA vs. SYNCRIP siRNA; 5) Differntially spliced genes in ctrl siRNA vs. SYNCRIP siRNA; 6) Differentially spliced exons in ctrl siRNA vs. SYNCRIP siRNA
Project description:T cell activation leads to dramatic changes in cellular phenotype. We used CD3/CD28-activated human CD4 T cells to study how RNA binding proteins define the post-transcriptional landscape. Using RIPseq, we identified the RNA interactome of U2AF2 and show at the global level that U2AF2 binds the majority of transcripts that are differentially expressed and/or alternatively spliced during CD4 T cell activation. A unique protein interactome centered on U2AF2 is assembled in response to activation. Knocking down specific U2AF2 interacting partners (U2AF1, SYNCRIP, SRRM2, ILF2) selectively affects cytokine secretion and expression of activation markers. Furthermore, the expression and/or alternative splicing of transcripts important for immune cell function are also affected by knocking down these U2AF2 interacting proteins. U2AF1 and SYNCRIP knockdowns affect the proteins and transcripts bound to U2AF2, altering the transcriptome. Our work highlights the importance of RNA binding protein complexes in regulating the differential expression and alternative splicing that defines T cell activation. HTA 2.0 microarray results of total from a primary CD4 T cell culture after treatment with siRNAs (Control, U2AF1, SRRM2, SYNCRIP, and ILF2) and 24 hours after anti-CD3/CD28 bead activation Please note that the Series supplementary 'TotalRNA_HTA2_all_siRNA_100414.xslx' file has multiple worksheets containing: 1) Differentially expressed genes in ctrl siRNA vs. U2AF1 siRNA; 2) Differntially spliced genes in ctrl siRNA vs. U2AF1 siRNA; 3) Differentially spliced exons in ctrl siRNA vs. U2AF1 siRNA; 4) Differentially expressed genes in ctrl siRNA vs. SRRM2 siRNA; 5) Differntially spliced genes in ctrl siRNA vs. SRRM2 siRNA; 6) Differentially spliced exons in ctrl siRNA vs. SRRM2 siRNA; 7) Differentially expressed genes in ctrl siRNA vs. SYNCRIP siRNA; 8) Differntially spliced genes in ctrl siRNA vs. SYNCRIP siRNA; 9) Differentially spliced exons in ctrl siRNA vs. SYNCRIP siRNA; 10) Differentially expressed genes in ctrl siRNA vs. ILF2 siRNA; 11) Differntially spliced genes in ctrl siRNA vs. ILF2 siRNA; 12) Differentially spliced exons in ctrl siRNA vs. ILF2 siRNA
Project description:T cell activation leads to dramatic changes in cellular phenotype. We used CD3/CD28-activated human CD4 T cells to study how RNA binding proteins define the post-transcriptional landscape. Using RIPseq, we identified the RNA interactome of U2AF2 and show at the global level that U2AF2 binds the majority of transcripts that are differentially expressed and/or alternatively spliced during CD4 T cell activation. A unique protein interactome centered on U2AF2 is assembled in response to activation. Knocking down specific U2AF2 interacting partners (U2AF1, SYNCRIP, SRRM2, ILF2) selectively affects cytokine secretion and expression of activation markers. Furthermore, the expression and/or alternative splicing of transcripts important for immune cell function are also affected by knocking down these U2AF2 interacting proteins. U2AF1 and SYNCRIP knockdowns affect the proteins and transcripts bound to U2AF2, altering the transcriptome. Our work highlights the importance of RNA binding protein complexes in regulating the differential expression and alternative splicing that defines T cell activation. RNAseq of total RNA from a primary CD4 T cell culture at rest and 48 hours after anti-CD3/CD28 bead activation
Project description:T cell activation leads to dramatic changes in cellular phenotype. We used CD3/CD28-activated human CD4 T cells to study how RNA binding proteins define the post-transcriptional landscape. Using RIPseq, we identified the RNA interactome of U2AF2 and show at the global level that U2AF2 binds the majority of transcripts that are differentially expressed and/or alternatively spliced during CD4 T cell activation. A unique protein interactome centered on U2AF2 is assembled in response to activation. Knocking down specific U2AF2 interacting partners (U2AF1, SYNCRIP, SRRM2, ILF2) selectively affects cytokine secretion and expression of activation markers. Furthermore, the expression and/or alternative splicing of transcripts important for immune cell function are also affected by knocking down these U2AF2 interacting proteins. U2AF1 and SYNCRIP knockdowns affect the proteins and transcripts bound to U2AF2, altering the transcriptome. Our work highlights the importance of RNA binding protein complexes in regulating the differential expression and alternative splicing that defines T cell activation. U2AF2 RIPseq on a primary CD4 T cell culture at rest and 48 hours after anti-CD3/CD28 bead activation
Project description:Despite clear evidence that exosomal microRNAs (miRNAs) are able to modulate the cellular microenvironment and that exosomal RNA cargo selection is often deregulated in pathological conditions, the mechanisms controlling specific RNA sorting into extracellular vescicles are still poorly understood. We identified here the RNA binding protein SYNCRIP (Synaptotagmin-binding Cytoplasmic RNA-Interacting Protein, also known as hnRNP-Q or NSAP1) as a component of the hepatocyte exosomal miRNA sorting machinery. SYNCRIP was found to bind directly a subset of miRNAs enriched in exosomes, sharing a common extra-seed sequence that we named hEXO motif. In vivo knock-down of SYNCRIP impaired microRNA sorting in exosomes and the hEXO motif was proven to play a role in the regulation of miRNA localization, as its embedment into a poorly exported miRNA enhanced its loading into exosomes. These results provide a new insight in the mechanisms of miRNA exosomal sorting process, that ca be exploited to further understand the role of these extracellular vescicles in cell-to-cell communications and the control of tissue micoenvironments.
Project description:Formaldehyde crosslinking and Proximity-dependent biotinylation methods were applied to study the cytoplasmic capping enzyme interactome via 1D-LC-MS/MS. The raw data were searched against Uniprot human database modified to contain our designed protein sequences using the Thermo Proteome Discoverer software (v 1.4.1.14).
Project description:SYNCRIP depletion results in HSCs losing their self renewal abilities. Single cell sequencing was conducted in SYNCRIP WT and KO bone marrow LK (lineage -, cKit+) cells to decipher the effect of Syncrip deletion on cellular identities along the hematopoietic hierarchy, and to gain an in-depth assessment of the transcriptomic changes in different cell types upon SYNCRIP loss.
Project description:During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15 kb 3’ untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the mRNA stability of the long prosperoisoform, which allows an upregulation of Prospero protein production. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.
Project description:Oocytes must accumulate and store maternal factors such as proteins during growth to sustain the first stages of embryonic development. How mammalian oocytes store maternal proteins is not understood. Here, we show that mouse and human oocytes store proteins on cytoplasmic lattices. With super-resolution light microscopy and electron tomography, we demonstrate that cytoplasmic lattices are twisted filament bundles formed by proteins of the subcortical maternal complex, filling the entire ooplasm. The lattices were associated with many proteins that have crucial functions during early embryo development, including proteins controlling genome methylation. Loss of cytoplasmic lattices prevented the accumulation of these proteins and resulted in early embryonic arrest. Thus, cytoplasmic lattices are important for storage of essential maternal proteins and the developmental success of early mammalian embryos.