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Small-RNA asymmetry is directly driven by mammalian Argonautes

ABSTRACT: Asymmetric selection of single-stranded guide RNAs from double-stranded RNA (dsRNA) precursors is crucial for RNA silencing-mediated gene regulation. However, the precise mechanisms for small RNA asymmetry remain unclear, especially since asymmetric selection can still occur under depletion of putative asymmetry sensors, Drosophila R2D2 and mammalian Dicer. Here we report direct contribution of mammalian Argonaute 2 (Ago2) to microRNA (miRNA) asymmetry. Ago2 selects strands with 5´-uridine/adenosine and thermodynamically unstable 5´-ends in parallel through its two sensor regions, which contact 5´-nucleobase and 5´-phosphate(s) of the prospective guide strands, respectively. Consistently, miRNA asymmetry shows characteristic digital-analog superposed patterns reflecting 5'-end nucleotide identity and thermodynamic stability. Furthermore, we demonstrate that cancer-associated miRNA variations reprogram asymmetric selection. Finally, our study presents a model of this universal principle that will aid a comprehensive understanding of miRNA function and therapeutic reinvention of RNA silencing in precision medicine.

ORGANISM(S): Mus musculus

PROVIDER: GSE66022 | GEO | 2015/06/15

SECONDARY ACCESSION(S): PRJNA275702

REPOSITORIES: GEO

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Small-RNA asymmetry is directly driven by mammalian Argonautes

Project description:Asymmetric selection of single-stranded guide RNAs from double-stranded RNA (dsRNA) precursors is crucial for RNA silencing-mediated gene regulation. However, the precise mechanisms for small RNA asymmetry remain unclear, especially since asymmetric selection can still occur under depletion of putative asymmetry sensors, Drosophila R2D2 and mammalian Dicer. Here we report direct contribution of mammalian Argonaute 2 (Ago2) to microRNA (miRNA) asymmetry. Ago2 selects strands with 5´-uridine/adenosine and thermodynamically unstable 5´-ends in parallel through its two sensor regions, which contact 5´-nucleobase and 5´-phosphate(s) of the prospective guide strands, respectively. Consistently, miRNA asymmetry shows characteristic digital-analog superposed patterns reflecting 5'-end nucleotide identity and thermodynamic stability. Furthermore, we demonstrate that cancer-associated miRNA variations reprogram asymmetric selection. Finally, our study presents a model of this universal principle that will aid a comprehensive understanding of miRNA function and therapeutic reinvention of RNA silencing in precision medicine. Immunoprecipitation of WT or mutant Ago2 in mouse ES cells (Ago-null background) and small RNA sequencing

2015-06-15 | E-GEOD-66022 | biostudies-arrayexpress

siRNA profiling of HeLa cells reveals strand-specific 5-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity.

Project description:Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity. Experiment Overall Design: For details please see: Chen PY, Weinmann L, Gaidatzis D, Pei Y, Zavolan M, Tuschl T, Meister G. RNA (2008) 14(2):263-74.

2008-06-11 | E-GEOD-10960 | biostudies-arrayexpress

Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity.

Project description:Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity. Keywords: siRNA, transfection, chemical modification, off-targets

2008-04-02 | GSE10960 | GEO

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

Project description:In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded characterM-bM-^@M-^Tas is found in small interfering RNAs (siRNAs)M-bM-^@M-^Tdirects duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide. Sequencing of small RNAs (either total small RNAs or Ago1-associated small RNAs) in wild-type, dcr-2 and r2d2 mutant flies. Small RNA sequencing, Small RNAs (18-29 nt long), Size selection (18 to 30 nt).

2009-10-31 | E-GEOD-18806 | biostudies-arrayexpress

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

Project description:In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.

2009-10-31 | GSE18806 | GEO

Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans

Project description:Many Argonaute proteins can cleave RNA (“slicing”) as part of the microRNA-induced silencing complex (miRISC), even though miRNA-mediated target repression is generally independent of target cleavage. Here we use C. elegans to examine the role of catalytic residues of miRNA Argonautes in organismal development. In contrast to previous work, mutations in presumed catalytic residues did not interfere with normal development when introduced by CRISPR. We find that unwinding and decay of miRNA star strands is weakly defective in the catalytic residue mutants, with the largest effect observed in embryos. Argonaute-Like Gene 2 (ALG-2) is more dependent on slicing for unwinding than ALG-1. The miRNAs that displayed the greatest (albeit minor) dependence on catalytic residues for unwinding tend to form stable duplexes with their star strand, and in some cases, lowering duplex stability alleviates dependence on catalytic residues. While a few miRNA guide strands are reduced in the mutant background, the basis of this is unclear since changes were not dependent on EBAX-1, an effector of Target-Directed miRNA Degradation (TDMD). Overall, this work defines a role for the catalytic residues of miRNA Argonautes in star strand decay; future work should examine whether this role may contribute to the selection pressure to conserve catalytic activity of miRNA Argonautes across the metazoan phylogeny.

2024-02-26 | GSE256488 | GEO

Catalytic residues of microRNA Argonautes play a modest role in microRNA star strand destabilization in C. elegans [miRNA]

2024-02-26 | GSE256489 | GEO

Nematostella vectensis argonautes

Project description:We performed Immuno precipitation using antibodies against Nematostella vectensis Argonauts proteins (i.e AGO1 and AGO2). Samples of the IP were analyzed by LC-MSMS in order to idetify the argonautes and prove antibodies specifity.

2020-10-14 | PXD011644 | Pride

Hierarchical rules for Argonaute loading in Drosophila

Project description:Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. microRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a by-product of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway. These were independently processed and sequenced using the Illumina GAII platform. In total, five libraries were analyzed.

2009-11-15 | E-GEOD-17734 | biostudies-arrayexpress

Asymmetric distribution of H3K9me3 at replicating DNA strands silences L1 retrotransposons during S phase

Project description:In higher eukaryotes, repetitive DNA sequences are transcriptionally silenced through histone H3 lysine 9 tri-methylation (H3K9me3). A loss of silencing of these elements leads to genome instability and human diseases, including cancer and aging1-3. While the role of H3K9me3 in the establishment and maintenance of heterochromatin and repression of DNA repeats has been extensively studied4-6, the pattern and mechanism underlying the partitioning of H3K9me3 at replicating DNA strands were unknown. Here, we report that H3K9me3 is preferentially transferred onto the leading strands of replication forks, which occurs predominantly at Long Interspersed Nuclear Element (LINE) retrotransposons that are theoretically transcribed at the head-on direction with replication fork movement. In contrast, all other modified forms of histones tested are distributed in a nearly symmetric manner at the leading and lagging strands. Mechanistically, the Human Silencing Hub (HUSH) complex interacts with the leading strand DNA polymerase Pol ε, and contributes to the asymmetric segregation of H3K9me3 during chromatin replication. Cells deficient for Pol ε (POLE3 and POLE4), the HUSH complex (MPP8 and TASOR), or cells expressing a MPP8 mutant defective of H3K9me3 binding, or TASOR mutants with reduced interaction with Pol ε, show compromised H3K9me3 asymmetry and increased LINE expression. These results reveal an unexpected mechanism whereby the HUSH complex functions with Pol ε to promote asymmetric H3K9me3 distribution at LINEs to suppress their expression during S phase.

2023-08-17 | GSE211192 | GEO

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