Project description:The aim of the study is to identify miRNA specific targets in Hodgkin lymphoma cell lines. By immunoprecipitation (IP) of wild type Ago2, Ago2 associated gene transcripts (ie miRNA targets) are coimmunoprecipitated. In cells transfected with anti-miR-17/20/93/106, the miR-17 seed family specific targets are not coimmunoprecipitated with Ago2. With microarray analysis, signal intensities of probes associated with Ago2 from untransfected and anti-miRNA transfected cells are compared. Gene transcripts that are depleted from the Ago2-IP fraction upon miRNA inhibition (ie miRNA specific targets) are identified.
Project description:MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by inhibiting protein synthesis of target messenger RNAs (mRNAs). MicroRNA-142 (miR-142), which has tumor-suppressive properties, was functionally deleted by CRISPR/Cas9 knockout in cell lines derived from diffuse large B-cell lymphoma (DLBCL), a highly aggressive tumor that represents about 30% of non-Hodgkin lymphoma worldwide. Mutations in miR-142 affect about 20% of all cases of DLBCL. By proteome analyses, the miR-142 knockout resulted in a consistent up-regulation of 52 but also down-regulation of 41 proteins in the GC-DLBCL lines BJAB and SUDHL4. Various mitochondrial ribosomal proteins were up-regulated in line with their pro-tumorigenic properties, while proteins necessary for MHC-I presentation were down-regulated in accordance with the finding that miR-142 knockout mice have a defective immune response. Of the deregulated proteins/genes, CFL2, CLIC4, STAU1, and TWF1 are known targets of miR-142, and we could additionally confirm AKT1S1, CCNB1, LIMA1, and TFRC as new targets of miR-142-3p or -5p. We further show that seed-sequence mutations of miR-142 can be used to confirm potential targets and that miRNA knockout cell lines might thus be used to identify novel targets of miRNAs. Due to the complex contribution of miRNAs within cellular regulatory networks, in particular when a miRNA highly present in the RISC complex is deleted and can be replaced by other endogenous miRNAs, primary effects on gene expression may be covered by secondary layers of regulation
Project description:In Burkitt lymphoma (BL), a network consisting of MYC, MYC-repressed miR-150, known miR-150 target MYB and two novel targets of miR-150, ZDHHC11 and ZDHHC11B, has been established. This network plays an important role on the growth of BL cells. Here, we confirmed that MYB, ZDHHC11 and ZDHHC11B are targeted by miR-150 in Hodgkin lymphoma (HL) cell lines too.
Project description:miR-17-92 mediates the MYC oncogene addiction in a conditional mouse lymphoma model. To identify targets of miR-17-92 in this model, miR-17-92 was expressed in the conditional lymphoma cell lines using MSCV-puro.
Project description:miR-17-92 mediates the MYC oncogene addiction in a conditional mouse lymphoma model. To identify targets of miR-17-92 in this model, miR-17-92 was expressed in the conditional lymphoma cell lines using MSCV-puro. Both control and miR-17-92-expressing conditional lymphoma cell lines were treated with doxycycline (DOX) (20ng/ml) for 48 hours to shut off MYC expression.
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Two replicates of two cDNA libraries were submitted to deep sequencing: a sample from siH19-transfected cells and a control sample.
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Two replicates of three cDNA libraries were submitted to deep sequencing: a sample from RNA-7-transfected cells; a sample from pre-miR-106a transfected cells; and a control sample.
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs.
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs.
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs.