Genomic basis of a social polymorphism in a halictid bee

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The Cell Cycle Regulator CCDC6 Is a Key Target of RNA-Binding Protein EWS

<div><p>Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma.</p></div

Regulation of targets by EWS in vivo.

<p><b>A</b>. Protein domain organization of EWS and FLI1. The black vertical arrows indicate common breakpoints in Ewing sarcoma. Numbers correspond to exons and a typical EWS-FLI1 fusion protein is also shown. Note that the RNA-binding domain of EWS is lost in the process of translocation. <b>B</b>. Pie diagram showing the distribution of PAR-CLIP clusters across 3’UTR, 5’UTR, intronic and coding regions of Refseq RNAs. The three diagrams give the cluster distribution of all sequenced EWS PAR-CLIP targets, all targets regulated by EWS and the four targets we validated (FGF9, MDM2, CBFB, CCDC6). <b>C</b>. Relative mRNA levels of targets genes FGF9, MDM2, CBFB, CCDC6 and EWS in HEK293T cells following EWS knockdown assayed by qRT-PCR (mock: only transfection reagent used; scrambled: AllStars Negative Control siRNA; EWS: siRNA targeting EWS). Relative mRNA levels were normalized to beta actin and quantified relative to the mock and scrambled control levels. Results are shown as mean SEM values (*P < 0.05; n = 3 per group). <b>D</b>. Amount of CCDC6 mRNA transcript percentage is measured upon knocking down of EWS as compared to control. The level of transcript was measured by qRT-PCR after knocking down for 24 hours followed by treatment with actinomycin D. The linear regression and slopes were calculated and the data is presented as Mean and SEM on a linear scale. <b>E</b>. Luciferase activity of CCDC6 upon EWS transfection (normalized to empty psiCHECK-2 plasmid). Data is shown as the fold increase in luciferase activity (RLU units) relative to control. Results are shown as the mean SEM values (*P<0.05; n = 3 per group). <b>F</b>. Relative mRNA levels of CCDC6 and EWS in mock, control and EWS knockdown in MHH-ES-1 cells. Knockdown of EWS decreased the expression of CCDC6. The mRNA levels were normalized to beta actin. Data is represented as mean SEM values (*P<0.05; n = 3 per group). <b>G</b>. Western blot showing the downregulation of CCDC6 upon EWS knockdown in MHH-ES-1 cells. Antibodies are indicated.</p

YBX1 Indirectly Targets Heterochromatin-Repressed Inflammatory Response-Related Apoptosis Genes through Regulating CBX5 mRNA.

Medulloblastomas arise from undifferentiated precursor cells in the cerebellum and account for about 20% of all solid brain tumors during childhood; standard therapies include radiation and chemotherapy, which oftentimes come with severe impairment of the cognitive development of the young patients. Here, we show that the posttranscriptional regulator Y-box binding protein 1 (YBX1), a DNA- and RNA-binding protein, acts as an oncogene in medulloblastomas by regulating cellular survival and apoptosis. We observed different cellular responses upon YBX1 knockdown in several medulloblastoma cell lines, with significantly altered transcription and subsequent apoptosis rates. Mechanistically, PAR-CLIP for YBX1 and integration with RNA-Seq data uncovered direct posttranscriptional control of the heterochromatin-associated gene CBX5; upon YBX1 knockdown and subsequent CBX5 mRNA instability, heterochromatin-regulated genes involved in inflammatory response, apoptosis and death receptor signaling were de-repressed. Thus, YBX1 acts as an oncogene in medulloblastoma through indirect transcriptional regulation of inflammatory genes regulating apoptosis and represents a promising novel therapeutic target in this tumor entity

Transcriptome-wide analysis uncovers the targets of the RNA-binding protein MSI2 and effects of MSI2's RNA-binding activity on IL-6 signaling

The RNA-binding protein Musashi 2 (MSI2) has emerged as an important regulator in cancer initiation, progression, and drug resistance. Translocations and deregulation of the MSI2 gene are diagnostic of certain cancers, including chronic myeloid leukemia (CML) with translocation t(7;17), acute myeloid leukemia (AML) with translocation t(10;17), and some cases of B-precursor acute lymphoblastic leukemia (pB-ALL). To better understand the function of MSI2 in leukemia, the mRNA targets that are bound and regulated by MSI2 and their MSI2-binding motifs need to be identified. To this end, using photoactivatable ribonucleoside cross-linking and immunoprecipitation (PAR-CLIP) and the Multiple EM for Motif Elicitation (MEME) analysis tool, here we identified MSI2’s mRNA targets and the consensus RNA-recognition element (RRE) motif recognized by MSI2 (UUAG). Of note, MSI2 knockdown altered the expression of several genes with roles in eukaryotic initiation factor 2 (eIF2), hepatocyte growth factor (HGF), and epidermal growth factor (EGF) signaling pathways. We also show that MSI2 regulates classic interleukin-6 (IL-6) signaling by promoting the degradation of the mRNA of IL-6 signal transducer (IL6ST or GP130), which, in turn, affected the phosphorylation statuses of signal transducer and activator of transcription 3 (STAT3) and the mitogen-activated protein kinase ERK. In summary, we have identified multiple MSI2-regulated mRNAs and provided evidence that MSI2 controls IL6ST activity that control oncogenic signaling networks. Our findings may help inform strategies for unraveling the role of MSI2 in leukemia to pave the way for the development of targeted therapies