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Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015)

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Homo sapiens

PROVIDER: GSE75802 | GEO | 2015/12/09

SECONDARY ACCESSION(S): PRJNA305401

REPOSITORIES: GEO

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Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015)

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2015-12-09 | E-GEOD-75802 | biostudies-arrayexpress

Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner.

Project description:MicroRNAs are short (17-26) noncoding RNAs driving or modulating physiological and pathological cellular events. Overexpression of miR-155 is pathogenic in B-cell malignancy but was also reported in a number of solid tumors-in particular, in breast cancer, where its role remains unclear and often contradictory. Using representative cell line models, we sought to determine whether the discrepant miR-155 effects in breast cancer could be explained by the heterogeneity of the disease. The growth of six breast cancer cell lines transfected with several miRNA mimics was analyzed. We found MCF-7 cell growth to be inhibited by miR-155 and miR-145 mimics, both 23-nt long, but not by a number of shorter mimics, including a universal commercial negative control. Microarray and Western blot analyses revealed induction of apoptosis, associated with interferon-β after activation of the double-stranded RNA sensor pathway. 3' Trimming of the miRNA mimics to 21 nt substantially reduced their growth-inhibitory potency. Mutating the canonical seed of the miR-155 mimic had no effect on the induced inhibition, which was abolished by mutating the miRNA seed of the artificial passenger strand. A panel of breast cancer cell lines showed a wide range of sensitivities to 23-mer mimics, broadly consistent with the sensitivity of the cell lines to Poly (I:C). We demonstrate two sources for nonspecific in vitro effects by miRNA mimics: duplex length and the artificial passenger strand. We highlight the danger of a universal 21-mer negative control and the importance of using matched seed mutants for reliable interpretation of phenotypes.

| S-EPMC4712670 | biostudies-literature

Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015), Exp 1

Project description:Experiment 1 - miR-155 and miR-199 Phenotype miR-155 and miR-199 as 23mer miRNA mimics induced a double-stranded RNA response Cells were transfected with miR-155, miR-199 23 mers, negative control or nothing (empty); samples collected in triplicate

2015-12-09 | E-GEOD-75800 | biostudies-arrayexpress

Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015), Exp 2

Project description:Experiment 2 - MiRNA mimics have a length and passenger strand specific effect Wildtype and mutant variants of miR-155 and negative control miRNA mimics were transfected to address the phenotype Cells were transfected with mimics and samples were collected in duplicate (except 23mer miR-155 wild type mimic and 23mer negative control mimic)

2015-12-09 | E-GEOD-75801 | biostudies-arrayexpress

Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015), Exp 2

2015-12-09 | GSE75801 | GEO

Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015), Exp 1

Project description:Experiment 1 - miR-155 and miR-199 Phenotype miR-155 and miR-199 as 23mer miRNA mimics induced a double-stranded RNA response

2015-12-09 | GSE75800 | GEO

Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015)

Project description:Double-stranded microRNA mimics can induce length- and passenger strand-dependent effects in a cell type-specific manner (RNA 2015)

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Passenger strand loading in overexpression experiments using microRNA mimics.

Project description:MicroRNAs (miRNAs) are important regulators of gene function and manipulation of miRNAs is a central component of basic research. Modulation of gene expression by miRNA gain-of-function can be based on different approaches including transfection with miRNA mimics; artificial, chemically modified miRNA-like small RNAs. These molecules are intended to mimic the function of a miRNA guide strand while bypassing the maturation steps of endogenous miRNAs. Due to easy accessibility through commercial providers this approach has gained popularity, and accuracy is often assumed without prior independent testing. Our in silico analysis of over-represented sequence motifs in microarray expression data and sequencing of AGO-associated small RNAs indicate, however, that miRNA mimics may be associated with considerable side-effects due to the unwanted activity of the miRNA mimic complementary strand.

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DNA double-strand breaks induce H2Ax phosphorylation domains in a contact-dependent manner.

Project description:Efficient repair of DNA double-strand breaks (DSBs) requires a coordinated DNA Damage Response (DDR), which includes phosphorylation of histone H2Ax, forming ?H2Ax. This histone modification spreads beyond the DSB into neighboring chromatin, generating a DDR platform that protects against end disassociation and degradation, minimizing chromosomal rearrangements. However, mechanisms that determine the breadth and intensity of ?H2Ax domains remain unclear. Here, we show that chromosomal contacts of a DSB site are the primary determinants for ?H2Ax landscapes. DSBs that disrupt a topological border permit extension of ?H2Ax domains into both adjacent compartments. In contrast, DSBs near a border produce highly asymmetric DDR platforms, with ?H2Ax nearly absent from one broken end. Collectively, our findings lend insights into a basic DNA repair mechanism and how the precise location of a DSB may influence genome integrity.

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Argonaute-3 activates the let-7a passenger strand microRNA.

Project description:MicroRNA duplices are separated into a guide and a passenger strand. By convention, the guide represents the active microRNA while the passenger is supposedly degraded. However, passenger strands also emerge as active microRNAs. It is unknown whether the guide-to-passenger-strand ratio can be actively regulated and which factors influence strand incorporation into the RISC. Here, we identify a microRNA with a variable guide-to-passenger-strand ratio along with its regulatory factor: Human Argonaute-3 specifically enhances the passenger strand expression and activity of the tumor suppressor microRNA let-7a. This post-maturational effect is mediated by the Ago3 PAZ and MID domains yielding an elevated affinity for let-7a-3p. Notably, this is independent of the 5'-terminal basepair stability, challenging the universality of the respective rule for microRNA strand selection. Thus, this study uncovers the first protein regulator of the ratio between microRNA guide and passenger strand expression and activity.

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