Identification of microRNA-668 and microRNA-489 targets by RISC immunoprecipitation
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE117805 | GEO | 2019/07/27
REPOSITORIES: GEO
Similar Datasets
Project description:Identification of microRNA-668 and microRNA-489 targets by RISC immunoprecipitation
| PRJNA483230 | ENA
Project description:It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function. This series includes microRNA sequencing data for human cell lines 293, C8166, A549, SH-SY5Y, and an EBV-transformed LCL line. For each cell line, total small RNA isolated by TRIzol was sequenced for comparison to RISC-associated microRNAs as determined by sequencing small RNAs from an Argonaute immunoprecipitation.
2014-05-30 | E-GEOD-58127 | biostudies-arrayexpress
Project description:293 cells were tranfected with Myc-tagged argonaute family members and associated microRNAs quantitated by microarray analysis of immunoprecipitates. Human argonaute 1, 2, and 3, and a control immunoprecipitation were done. Two independent immunoprecipitations were performed, with two replicate points per immunoprecipitation. Ch1and Ch2 net values are median intensity, background subtracted, unnormalized. Normalized Ch2 values for argonaute immunoprecipitations are median center normalized. Normalized Ch2 values for control immunoprecipitates are normalized by a constant that is the average of the constants used for argonaute immunoprecipitates. Keywords = microRNA Keywords = miRNA Keywords = argonaute Keywords = RNAi Keywords: repeat sample
2004-07-30 | GSE1573 | GEO
Project description:MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The mechanistic steps of miRNA biogenesis are well understood at molecular level1,2. Subsequent miRNA duplex loading into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing3-5. The Integrator complex has been previously identified as important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination, processes dependent on Integrator’s catalytical activity6-9. Here we report that absence of the Integrator complex leads to a global loss of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Indeed, Integrator enhances miRNA-Ago2 interaction in vitro, which functionally translates to amplified miRNA target cleavage. These findings demonstrate for the first time cytoplasmic Integrator complex functions and stress an indirect role of Integrator in transcription regulation through modulation of miRNA abundance and stability.
2023-02-14 | GSE178127 | GEO
Project description:293 cells were tranfected with Myc-tagged argonaute family members and associated microRNAs quantitated by microarray analysis of immunoprecipitates. Human argonaute 1, 2, and 3, and a control immunoprecipitation were done. Two independent immunoprecipitations were performed, with two replicate points per immunoprecipitation. Ch1and Ch2 net values are median intensity, background subtracted, unnormalized. Normalized Ch2 values for argonaute immunoprecipitations are median center normalized. Normalized Ch2 values for control immunoprecipitates are normalized by a constant that is the average of the constants used for argonaute immunoprecipitates. Keywords = microRNA Keywords = miRNA Keywords = argonaute Keywords = RNAi Keywords: repeat sample
2010-10-08 | E-GEOD-1573 | biostudies-arrayexpress
Project description:RNA from primary hepatocytes from pooled or individual donors was crosslinked to microRNA and Argonaute and pulled down for sequencing analysis.
2023-04-07 | GSE227405 | GEO
Project description:ARGONAUTE1 (AGO1) binds directly to small regulatory RNA and is a key effector protein of post-transcriptional gene silencing mediated by microRNA (miRNA) and small interfering RNA (siRNA). The formation of an RNA induced silencing complex (RISC) of AGO1 and small RNA requires the function of the Heat Shock Protein 70/90 chaperone system. Some functions of AGO1 occur in association with endomembranes, in particular the rough endoplasmic reticulum (rER), but proteins interacting with AGO1 in membrane fractions remain unidentified. In this study, we show that the farnesylated Heat Shock Protein 40 homologs, J2 and J3, associate with AGO1 in membrane fractions in a manner that involves protein farnesylation. We also show that three changes in AGO1 function are detectable in mutants in protein farnesylation and J2/J3. First, perturbations of the HSP40/70/90 pathway by mutation of j3, hsp90 and farnesyl transferase affect the amounts of AGO1 associated with membranes. Second, miRNA association with membrane-bound AGO1, and with membrane-bound polysomes is increased in farnesyl transferase and farnesylationdeficient J2/J3 mutants. Third, silencing by non-cell autonomously acting short interfering RNAs (siRNAs) is impaired. These observations highlight the involvement of farnesylated J2/J3 in small RNA-mediated gene regulation, and suggest that the importance of chaperone-AGO1 interaction is not limited to the RISC assembly process.
2018-09-10 | PXD010197 | Pride
Project description:Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics.
2012-07-12 | GSE39146 | GEO
Project description:MRJP-3 is an RNA binding protein that binds both double- and single-stranded RNA in vitro. To test whether i) MRJP-3 binds RNA in its natural environment, the royal jelly (RJ) and ii) to characterize its RJ RNA partners; a pull down followed by RNA-seq system was developed. MRJP-3 bound RNA was compared to total RJ RNA extracted from the same hive. To establish the system, we first incubated biotinylated MRJP-3, or biotinylated BSA in RJ and pulled the proteins out with strepavidin coated magnetic beads followed by RNA extraction and bioanalyzer profiling. MRJP-3 pull out was enriched for RNA compare to the BSA and just beads controls. The RNA population pulled down with MRJP-3 has similar profile to the total RJ RNA, further demonstrating the MRJP-3 has no affinity to a certain RNA species.
2018-10-28 | E-MTAB-6732 | biostudies-arrayexpress
Project description:We developed a proteomics approach to examine the mammalian CTD-interactome. We used six synthetic peptides each consisting of four consensus CTD-repeats and with different combinations of serine and tyrosine phosphorylation as affinity-matrix to pull-down nuclear proteins from HeLa cells. The pull-down fractions were then analyzed by MUDPIT mass spectrometry. This approach identified a total of 100 CTD-interacting proteins pull-downed by the differentially phosphorylated CTD-peptides. Our analyses showed that the majority of proteins pulled-down by serine-phosphorylatd CTD are involved in RNA processing. Furthermore, this study identified proteins that were preferentially pulled-down by tyrosine/serine-doubly phosphorylated CTD.
| MSV000078867 | MassIVE