Homo sapiens
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ABSTRACT:
PROVIDER: PRJNA107159 | ENA |
REPOSITORIES: ENA
Similar Datasets
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
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
Project description:microRNAs are M-bM-^HM-<22 nucleotide regulatory RNAs that are processed into duplexes from hairpin structures and incorporated into Argonaute proteins. We find that a nick in the middle of the guide strand of an miRNA sequence allows for seed-based targeting characteristic of miRNA activity. Insertion of an inverted abasic, a dye, or a small gap between the two segments still permits target knockdown. While activity from the seed region of the segmented miRNA is apparent, activity from the 3' half of the guide strand is impaired, suggesting that an intact guide backbone is required for contribution from the 3' half. miRNA activity was also observed following nicking of a miRNA precursor. These results illustrate a structural flexibility in miRNA duplexes and may have applications in the design of miRNA mimetics. For the microarray portion of the paper, a miR-124 duplex containing a segmented guide strand (G10.12/P) or an intact guide strand (G/P) was transfected into HCT-116 dicer- cells. Profiles were compared with mock transfections.
2010-11-12 | E-GEOD-25224 | biostudies-arrayexpress
Project description:microRNAs are ∼22 nucleotide regulatory RNAs that are processed into duplexes from hairpin structures and incorporated into Argonaute proteins. We find that a nick in the middle of the guide strand of an miRNA sequence allows for seed-based targeting characteristic of miRNA activity. Insertion of an inverted abasic, a dye, or a small gap between the two segments still permits target knockdown. While activity from the seed region of the segmented miRNA is apparent, activity from the 3' half of the guide strand is impaired, suggesting that an intact guide backbone is required for contribution from the 3' half. miRNA activity was also observed following nicking of a miRNA precursor. These results illustrate a structural flexibility in miRNA duplexes and may have applications in the design of miRNA mimetics.
2010-11-12 | GSE25224 | GEO
Project description:siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand ‘‘seed region,’’ similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models. Hepa1-6 mouse hepatoma cell line and HUH7 and PLC/PRF/5 human hepatoma cell lines were transfected in 6-well plates using Lipofectamine RNAiMAX and siRNA duplexes at a final concentration of 10 nM. For in vitro analysis, RNA was extracted at 6, 12, 24, and 48 h post-transfection. For in vivo studies, mouse livers were harvested 3 d following a single administration of APOB siRNA (3 mg/kg) formulated in lipid nanoparticles. For details, please see: J. Burchard, A.L. Jackson, V. Malkov, R.H.V. Needham, Y. Tan, S.R. Bartz, H. Dai, A.B. Sachs and P.S. Linsley. microRNA-like off-target transcript regulation by siRNAs is species specific. RNA 15 (2009)
2009-01-31 | E-GEOD-14073 | biostudies-arrayexpress
Project description:In plants, transcriptional silencing by RNA-directed DNA methylation (RdDM) is mediated by ARGONAUTE 4 (AGO4) and 24 nt short-interfering RNAs (siRNAs) that are generated in parallel with 23 nt RNAs of unknown function. We show that 23 nt RNAs serve as the passenger strands of 23/24 nt duplexes loaded into AGO4. The 24 nt siRNAs then guide AGO4 slicing of the passenger strands, generating 11 and 12 nt cleavage products. Unexpectedly, we find that the 12 nt products remain associated with the guide strand-AGO4 complexes. Long noncoding RNAs generated at RdDM loci are similarly sliced and retained by AGO4. These results suggest a model in which RNA POLYMERASE V transcripts at target loci are sliced repeatedly as transcription elongation proceeds, sequentially releasing AGO4-siRNA-scaffold RNA complexes that independently recruit the RdDM machinery. Consistent with this hypothesis, plant lines expressing wild-type versus slicing-defective AGO4 show quantitative variation in cytosine methylation and siRNA levels within RdDM loci.
2023-02-17 | GSE201233 | GEO
Project description:In plants, transcriptional silencing by RNA-directed DNA methylation (RdDM) is mediated by ARGONAUTE 4 (AGO4) and 24 nt short-interfering RNAs (siRNAs) that are generated in parallel with 23 nt RNAs of unknown function. We show that 23 nt RNAs serve as the passenger strands of 23/24 nt duplexes loaded into AGO4. The 24 nt siRNAs then guide AGO4 slicing of the passenger strands, generating 11 and 12 nt cleavage products. Unexpectedly, we find that the 12 nt products remain associated with the guide strand-AGO4 complexes. Long noncoding RNAs generated at RdDM loci are similarly sliced and retained by AGO4. These results suggest a model in which RNA POLYMERASE V transcripts at target loci are sliced repeatedly as transcription elongation proceeds, sequentially releasing AGO4-siRNA-scaffold RNA complexes that independently recruit the RdDM machinery. Consistent with this hypothesis, plant lines expressing wild-type versus slicing-defective AGO4 show quantitative variation in cytosine methylation and siRNA levels within RdDM loci.
2023-02-17 | GSE201234 | GEO
Project description:In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are central to sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nt small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation (RdDM) pathway. Here, we determined structure of a DCL3-pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated-A1 of the guide strand and the 1-nt 3′-overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated-end of the guide RNA and dual cleavage sites formed by the paired RNaseIII domains. These structural studies, complemented by functional data, reveal insights into the dicing principle for Dicers in general.
2021-10-14 | GSE179616 | GEO
Project description:Datasets from manuscript: Caenorhabditis elegans ALG-1 antimorphic mutations uncover functions for Argonaute in microRNA guide strand selection and passenger strand disposal
| MSV000079287 | MassIVE
Project description:To identify genes regulated by NFE2L2 (Nrf2), we selected a lung cancer cell line (A549) in which NFE2L2 is normally active. Three transfections using siRNAs targeting NFE2L2 and four control transfections using two different negative control siRNAs were done. As a result, we found several genes up or down regulated in response to NFE2L2 inactivation in these cells. A549 cells were cultured, and 250,000 cells were transfected with Lipofectamine RNAiMAX reagent containing 5 nmol siRNA particles in a 6-well plate. Three transfections with siRNAs targeting NFE2L2 were performed, using three independent duplexes, while four control transfections were done using two different control duplexes, in addition to one mock (Lipofectamine only) transfection. RNA was prepared using the RNeasy Mini kit. 8 strand-specific total RNA-seq libraries were generated on an Illumina NextSeq 500. Between 50.5 and 60.6 million reads were obtained per sample. Reads were aligned to hg19 genome, at an alignment rate exceeding 94.4% for all samples.
2016-10-25 | E-GEOD-75452 | biostudies-arrayexpress