Project description:Aberrant RNA-editing was observed in several human tumors, but its significance is mostly unknown. Here we show that ADAR1, a ubiquitous RNA-editing enzyme, is commonly lost in metastatic melanoma cells and specimens. Experimental ADAR1 silencing significantly alters melanoma cell morphology, facilitates proliferation and cell-cycle, and increases the tumorigenicity in-vivo. A series of ADAR1 truncation mutants establishes a novel RNA-editing-independent role for ADAR1 in controlling the nuclear and cytoplasmic processing steps of miRNA biogenesis. Altered expression of ADAR1-controled miRNAs accounts for the observed phenotype. We show that the oncogenic miR-17-5p endogenously regulates ADAR1 expression and that its genomic sequence is frequently amplified in melanoma to overexpress the mature miR-17-5p form. ADAR1 and miR-17-5p are ubiquitously expressed, suggesting the generality of this mechanism. Melanoma cell line expressing low ADAR1 levels (ADAR1-Knockdown) using shRNA technique were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to examine the alterations in the genes and microRNA expression profile in the manipulated cell system, due to ADAR1 possible involvement cancer development. To that end, we selected ADAR1-knockdown (ADAR1-KD) cells that demonstrated an enhanced aggressive phenotype both in vivo and in vitro as compared to the control cells (Control).
Project description:Aberrant RNA-editing was observed in several human tumors, but its significance is mostly unknown. Here we show that ADAR1, a ubiquitous RNA-editing enzyme, is commonly lost in metastatic melanoma cells and specimens. Experimental ADAR1 silencing significantly alters melanoma cell morphology, facilitates proliferation and cell-cycle, and increases the tumorigenicity in-vivo. A series of ADAR1 truncation mutants establishes a novel RNA-editing-independent role for ADAR1 in controlling the nuclear and cytoplasmic processing steps of miRNA biogenesis. Altered expression of ADAR1-controled miRNAs accounts for the observed phenotype. We show that the oncogenic miR-17-5p endogenously regulates ADAR1 expression and that its genomic sequence is frequently amplified in melanoma to overexpress the mature miR-17-5p form. ADAR1 and miR-17-5p are ubiquitously expressed, suggesting the generality of this mechanism. Melanoma cell line expressing low ADAR1 levels (ADAR1-Knockdown) using shRNA technique were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to examine the alterations in the genes and microRNA expression profile in the manipulated cell system, due to ADAR1 possible involvement cancer development. To that end, we selected ADAR1-knockdown (ADAR1-KD) cells that demonstrated an enhanced aggressive phenotype both in vivo and in vitro as compared to the control cells (Control).
Project description:TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription.
Project description:The differentiation of myoblasts plays a key role in the growth of biological individuals and the reconstruction of muscle tissue. Several microRNAs are significantly upregulated during the differentiation of myoblasts and their target genes have been explored. However, the molecular mechanisms underlying the transcriptional regulation of microRNAs remain elusive. In the present study, we found that the expression of miR-133a is increased during the differentiation of C2C12 myoblasts. miR-133a mimic is sufficient to induce the biogenesis of mitochondria and differentiation of C2C12 myoblasts whereas miR-133a inhibitor abolishes cell differentiation. Using CRISPR affinity purification in situ of regulatory elements (CAPTURE) technique, we further dissected the regulatory mechanisms of miR-133a expression and found that KAP1-associated transcription complex accounts for the suppression of miR-133a in C2C12 myoblasts. Knockdown of KAP1 increased the expression of miR-133a, which contributed to the biogenesis of mitochondria and differentiation of C2C12 myoblasts. To our knowledge, this is the first study using the CAPTURE technology to identify the regulatory factors of miR-133a during cell differentiation, which may provide new ideas for understanding the precision regulatory machinery of microRNAs during different biological processes.
Project description:Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by germline inactivating mutations of TSC1 or TSC2. In TSC-associated tumors of the brain, heart, skin, kidney and lung, inactivation of both alleles of TSC1 or TSC2 leads to hyperactivation of the mTORC1 pathway. The TSC/mTORC1 pathway is a key regulator of cellular processes related to growth, proliferation and autophagy. We and others have previously found that mTORC1 regulates microRNA biogenesis, but the mechanisms are not fully understood. Microprocessor, a multi-protein complex including the nuclease Drosha, processes the primary miR transcript. Using a dual-luciferase reporter, we found that inhibition of mTORC1 or downregulation of Raptor decreased Microprocessor activity, while loss of TSC2 led to a striking increase (?5-fold) in Microprocessor activity. To determine the global impact of TSC2 on microRNAs we quantitatively analyzed 752 microRNAs in Tsc2-expressing and Tsc2-deficient cells. Out of 259 microRNAs expressed in both cell lines, 137 were significantly upregulated and 24 were significantly downregulated in Tsc2-deficient cells, consistent with the increased Microprocessor activity. Microprocessor activity is known to be regulated in part by GSK3?. We found that total GSK3? levels were higher in Tsc2-deficient cells, and the increase in Microprocessor activity associated with Tsc2 loss was reversed by three different GSK3? inhibitors. Furthermore, mTOR inhibition increased the levels of phospho-GSK3? (S9), which negatively affects Microprocessor activity. Taken together these data reveal that TSC2 regulates microRNA biogenesis and Microprocessor activity via GSK3?.
Project description:TORC1 is a structurally and functionally conserved multiprotein complex that regulates many aspects of eukaryote growth including the synthesis and assembly of ribosomes. The protein kinase activity of this complex is responsive to environmental cues and is potently inhibited by the natural product macrolide rapamycin. Insights into how TORC1 regulates growth have been provided with the recent identification of the rapamycin-sensitive phosphoproteome in yeast. Building on these data, we show here that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of three transcription repressors, Stb3, Dot6 and Tod6. Dephosphorylation of these factors allows them to recruit the RPD3L histone deactelyase complex to ribi/RP gene promoters. Since rRNA and tRNA transcription are also under its control, Sch9 appears to be well positioned to coordinately regulate transcriptional aspects of ribosome biogenesis. mRNA-Seq of 8 S. cerevisiae strains treated with either DMSO alone or 1NM-PP1, a small molecule inhibitor for analog-sensitive kinases such as sch9-as.
Project description:TORC1 is a structurally and functionally conserved multiprotein complex that regulates many aspects of eukaryote growth including the synthesis and assembly of ribosomes. The protein kinase activity of this complex is responsive to environmental cues and is potently inhibited by the natural product macrolide rapamycin. Insights into how TORC1 regulates growth have been provided with the recent identification of the rapamycin-sensitive phosphoproteome in yeast. Building on these data, we show here that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of three transcription repressors, Stb3, Dot6 and Tod6. Dephosphorylation of these factors allows them to recruit the RPD3L histone deactelyase complex to ribi/RP gene promoters. Since rRNA and tRNA transcription are also under its control, Sch9 appears to be well positioned to coordinately regulate transcriptional aspects of ribosome biogenesis. ChIP-Seq of 8 S. cerevisiae strains treated with 1NM-PP1, a small molecule inhibitor for analog-sensitive kinases such as sch9-as.
Project description:WBP2 is an emerging oncoprotein with diverse functions in breast tumorigenesis via regulating Wnt, epidermal growth factor receptor, estrogen receptor, and Hippo. Recently, evidence shows that WBP2 is tightly regulated by the components of the miRNA biogenesis machinery such as DGCR8 and Dicer via producing both WBP2's 3'UTR and coding DNA sequence-targeting miRNAs. This led us to hypothesize that WBP2 could provide a feedback loop to the biogenesis of its key upstream regulators by regulating the microprocessor complex activity. Indeed, WBP2 suppressed microprocessor activity by blocking the processing of pri-miRNAs to pre-miRNAs. WBP2 negatively regulated the assembly of the microprocessor complex via physical interactions with its components. Meta-analyses suggest that microprocessor complex components, in particular DGCR8, DDX5, and DEAD-Box Helicase17 (DDX17), have tumor-suppressive properties. 2D and 3D in vitro proliferation assays revealed that WBP2 blocked the tumor-suppressive properties of DGCR8, a key component of the microprocessor complex. In conclusion, WBP2 is a novel regulator of miRNA biogenesis that is a known dysregulated pathway in breast tumorigenesis. The reregulation of miRNA biogenesis machinery via targeting WBP2 protein may have implications in breast cancer therapy.
Project description:Aberrant RNA-editing was observed in several human tumors, but its significance is mostly unknown. Here we show that ADAR1, a ubiquitous RNA-editing enzyme, is commonly lost in metastatic melanoma cells and specimens. Experimental ADAR1 silencing significantly alters melanoma cell morphology, facilitates proliferation and cell-cycle, and increases the tumorigenicity in-vivo. A series of ADAR1 truncation mutants establishes a novel RNA-editing-independent role for ADAR1 in controlling the nuclear and cytoplasmic processing steps of miRNA biogenesis. Altered expression of ADAR1-controled miRNAs accounts for the observed phenotype. We show that the oncogenic miR-17-5p endogenously regulates ADAR1 expression and that its genomic sequence is frequently amplified in melanoma to overexpress the mature miR-17-5p form. ADAR1 and miR-17-5p are ubiquitously expressed, suggesting the generality of this mechanism.