Global analyses of the effect of different cellular contexts on microRNA targeting (RNA-Seq)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Jin-Wu Nam
PROVIDER: E-GEOD-52530 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:RNA-seqs followed by miRNA transfections (miR-124 and miR-155) into four different cell lines( HeLa, HEK293, Huh7, and IMR90).
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Project description:MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We now demonstrate that endogenous and over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). We report that the "seed region" of moR-21 as well as the "seed match region" in the target gene 3'UTR are indispensable for moR-21-mediated gene down-regulation. We further demonstrated that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. Taken together, these findings provide the first evidence that microRNA offset RNA regulates gene expression and is biologically active. Small RNAs were harvested from mouse aortic smooth muscle cells (AoSMCs), with three biological replicates, using miReasy kits (Qiagen) according to the manufacturerâ??s instructions. RNA was quantified using a Nanodrop spectrophotometer. Small RNA library construction and illumina HiSeq 75bp single read sequencing were performed at the Yale Center for Genome Analysis. Reads were trimmed to remove adaptor sequences, and inserts longer than 13 bases were mapped to the mouse mm10 genome using Bowtie (with settings â??n 0 -m 5 --best --strata, allowing 0 mismatches and up to 5 genomic loci with a perfect match, {Genome Biol. 2009;10(3):R25, Langmead B, Trapnell C, Pop M, Salzberg SL}). Reads that mapped to within 50 bp of more than one annotated mouse pri-miR (as defined by miRBase {Kozomara A, Griffiths-Jones S. Nucelic Acids Res 2014 42:D68-D73}), were allotted evenly between these locations. Reads that fell entirely within established miRbase 3p or 5p miR coordinates, plus or minus 3 bp, were counted as 3p or 5p miRs, respectively. Reads that fell entirely within the range of 35 bp 3â?? to 3 bp 5â?? of the last base of a 3p miR were counted as 3p moR reads. Those that fell entirely between 3bp 3â?? and 35 bp 5â?? of the first base of a 5p miR were counted as 5p moR reads. On average each library contained 17.4 million qualifying inserts, of which 13.8 million (79.3%) mapped to one of these four regions, indicating that the large majority of inserts represented miR or moR sequences. Reads were divided by the number of million reads mapping to these regions in each library to give normalized reads per million reads (RPMR) values.
2016-06-01 | E-GEOD-75113 | biostudies-arrayexpress
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2010-06-25 | E-GEOD-22546 | biostudies-arrayexpress
Project description:We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr co-factor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its co-factors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs versus passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells. Series includes three datasets of total small RNA reads from wild type and Dicer negative 293T cells. Also included are total small RNA reads of the Dicer-negative cell line NoDice(4-25) transfected with a vector expressing human Dicer, Drosophila Dicer1, or mock transfected. RISC-associated small RNAs identified by ribonucleoprotein immunoprecipitation (RIP-Seq) in wild type 293T, Dicer-negative NoDice(4-25) line, and NoDice(4-25) transfected with either hsa-miR-155 or kshv-miR-K12-11 miRNA duplexes. The final data series are PAR-CLIP libraries which identified microRNA targets in untransfected NoDice(4-25) cells and NoDice(4-25) cells transfected with either hsa-miR-92a, hsa-miR-155, kshv-miR-K12-11 miRNA duplexes
2014-04-17 | E-GEOD-56836 | biostudies-arrayexpress
Project description:A network of gene regulatory factors such as transcription factors and microRNAs establish and maintain the gene expression pattern during hematopoiesis. In this network transcription factors regulate each other and are involved in regulatory loops with microRNAs.The microRNA cluster miR-17-92 is located within the MIR17HG gene and encodes for six mature microRNAs. It is important for hematopoietic differentiation and plays a central role in malignant disease. However, the transcription factors downstream of miR-17-92 are largely elusive and the transcriptional regulation of miR-17-92 is not fully understood. Here we show that miR-17-92 forms a regulatory loop with the transcription factor TAL1. The miR-17-92 cluster inhibits expression of TAL1 and indirectly leads to decreased stability of the TAL1 transcriptional complex. We found that TAL1 and its heterodimerization partner E47 regulate miR-17-92 transcriptionally. Furthermore, miR-17-92 negatively influences erythroid differentiation, a process that depends on gene activation by the TAL1 complex. Our data give example of how transcription factor activity is fine-tuned during normal hematopoiesis. We postulate that disturbance of the regulatory loop between TAL1 and the miR-17-92 cluster could be an important step in cancer development and progression.
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Project description:Brown adipose (BAT) development and function are unaffected by the absence of miR-193b/365-1 We refer to wild-type mice (+/+) as WT and to mice lacking the mir-193b (-/-) as Null mice.
2013-06-14 | E-GEOD-47926 | biostudies-arrayexpress