Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity

ABSTRACT: Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended (“off-target”) consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3’ UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence-mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with similar dependence on siRNA concentration as silencing of the target transcript. These results demonstrate that short stretches of sequence complementarity to the seed region of the siRNA are key to the silencing of unintended transcripts, and that this limits the specificity of siRNA-mediated transcript silencing. Because these off-target events are sequence-dependent, inclusion of multiple independent siRNAs to the target of interest can help to distinguish true positives from false positives in functional genetic analyses. Keywords: siRNA, RNAi, sequence alignment, off-target, seed region

ORGANISM(S): Homo sapiens

PROVIDER: GSE5814 | GEO | 2006/09/12

SECONDARY ACCESSION(S): PRJNA97197

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity

Project description:Transfected siRNAs and miRNAs regulate numerous transcripts that have only limited complementarity to the active strand of the RNA duplex. This process reflects natural target regulation by miRNAs, but is an unintended (â??off-targetâ??) consequence of siRNA-mediated silencing. Here we demonstrate that this unintended off-target silencing is widespread, and occurs in a manner reminiscent of target silencing by miRNAs. A high proportion of unintended transcripts silenced by siRNAs showed 3â?? UTR sequence complementarity to the seed region of the siRNA. Base mismatches within the siRNA seed region reduced the set of original off-target transcripts but generated new sets of silenced transcripts with sequence complementarity to the mismatched seed sequence. An inducible shRNA silenced a subset of transcripts that were silenced by an siRNA of the same sequence, demonstrating that unintended silencing is sequence-mediated and is independent of delivery method. In all cases, off-target transcript silencing was accompanied by loss of the corresponding protein and occurred with similar dependence on siRNA concentration as silencing of the target transcript. These results demonstrate that short stretches of sequence complementarity to the seed region of the siRNA are key to the silencing of unintended transcripts, and that this limits the specificity of siRNA-mediated transcript silencing. Because these off-target events are sequence-dependent, inclusion of multiple independent siRNAs to the target of interest can help to distinguish true positives from false positives in functional genetic analyses. For details, please see A.L. Jackson, et al. 2006. RNA 12(7): 1179-1187. We used consensus genelists for clustering. Please see attached tables for genelists.

2006-09-12 | E-GEOD-5814 | biostudies-arrayexpress

Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing

Project description:Transfected siRNAs regulate numerous transcripts sharing limited complementarity to the RNA duplex. This unintended (“off-target”) silencing can hinder the use of RNAi to define gene function. Here we describe position-specific, sequence-independent chemical modifications that reduced silencing of partially-complementary transcripts by all siRNAs tested. Silencing of perfectly-matched targets was unaffected by these modifications. The chemical modification also reduced off-target phenotypes in growth inhibition studies. Key to the modification was 2’-O-methyl ribosyl substitution at position 2 in the guide strand, which reduced silencing of most off-target transcripts with complementarity to the seed region of the siRNA guide strand. The sharp position-dependence of 2’-O-methyl ribosyl modification contrasts with the broader position dependence of base pair substitutions within the seed region, suggesting a role for position 2 of the guide strand distinct from its effects on pairing to target transcripts. Keywords: Microarray analysis, chemical modification walk, dose response

2006-09-07 | GSE5769 | GEO

microRNA-like off-target transcript regulation by siRNAs

2009-02-01 | GSE14073 | GEO

microRNA-like off-target transcript regulation by siRNAs

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

Reduced seed region-based off-target activity with lentivirus-mediated RNAi

Project description:In addition to silencing intended target genes, transfected siRNAs regulate numerous unintended transcripts through a mechanism in which the equivalent of a microRNA-like seed region in the siRNA recognizes complementary sequences in transcript 3' UTRs. The ability to limit such off-target effects would substantially facilitate accurate interpretation of RNA interference (RNAi) experiments and thus greatly enhance their value. We tested whether lentivirus-mediated delivery of shRNA is prone to the seed region-based off-target activity prevalent in siRNA experiments. We compared target gene silencing and overall impact on global gene expression caused by multiple 21-mer duplex sequences delivered as both transfected siRNA and lentivirus vector-expressed shRNA. At equivalent levels of target gene silencing, signatures induced by shRNAs were significantly smaller than those induced by cognate siRNAs and arose less frequently from seed region activity. Interestingly, the low level of seed-region based off-target activity exhibited by shRNAs resulted in down-regulation of transcripts that were largely distinct from those regulated by siRNAs. On the basis of these observations, we recommend lentivirus-mediated RNA interference for pathway profiling experiments that measure whole genome transcriptional readouts as well as for large-scale screens when resources for extensive follow up are limited.

2010-06-09 | GSE22209 | GEO

Homo sapiens

Project description:Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity

| PRJNA97197 | ENA

siRNAs targeting a chromatin-associated RNA induce its transcriptional silencing in human cells

Project description:Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, such as plants or fission yeasts. In mammals, its extent remained somewhat debated. Previous studies showed that siRNAs targeting gene promoters can induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by nascent RNA capture and RNA Pol II ChIP, we show that siRNAs targeting a chromatin-associated non-coding RNA induce its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further show that this silencing is due to base pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we show that only the wild type allele, but not the deleted allele, is silenced by the siRNA, indicating that transcriptional silencing occurs only in cis. Finally, we demonstrate that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs.

2022-11-18 | GSE197308 | GEO

Maternal siRNAs mediate spatial and temporal regulation of gene expression and seed development in Arabidopsis

Project description:In Arabidopsis seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the NRPD1 pathway. To investigate their biological functions we characterized siRNAs in the endosperm and seed coat that were separated by laser capture microdissection (LCM) in reciprocal genetic crosses with an nrpd1 mutant. We also monitored the spatio-temporal activity of the NRPD1 pathway on seed development using AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing. From these approaches we identified four distinct groups of siRNA loci that were dependent on or independent of the maternal NRPD1 allele in the endosperm or seed coat. A group of maternally expressed NRPD1-siRNA loci targets endosperm-preferred genes, including those encoding AGAMOUS-LIKE (AGL) transcription factors. Using the sensor AGL40:GUS and AGL91:GUS constructs we demonstrate that spatial and temporal expression patterns of these genes in the endosperm were regulated by the NRPD1-mediated pathway irrespective of complete silencing (AGL91) or incomplete silencing (AGL40) of these target genes. Modified expression of these siRNA-targeted genes affects seed size and we propose that the corresponding maternal siRNAs could account for parent-of-origin effects on the endosperm in interploidy and hybrid crosses. These analyses reconcile previous studies on siRNAs and imprinted gene expression during seed development.

2018-12-31 | GSE47722 | GEO

Systematic design and functional analysis of artificial microRNAs as an approach for multi-gene targeting

Project description:Unlike short interfering RNAs (siRNAs), which are commonly designed to repress a single messenger RNA (mRNA) target through perfect base pairing, microRNAs (miRNAs) are endogenous small RNAs that have evolved to concurrently repress multiple mRNA targets through imperfect complementarity. MicroRNA target recognition is primarily determined by pairing of the miRNA seed sequence (nucleotides 2–8) to complementary match sites in each mRNA target. Whereas siRNA technology is well established for single target knockdown, the design of artificial miRNAs for multi-target repression is largely unexplored. We designed and functionally analysed over 200 artificial miRNAs for simultaneous repression of pyruvate carboxylase and glutaminase by selecting all seed matches shared by their 3′ untranslated regions. Although we identified multiple miRNAs that repressed endogenous protein expression of both genes, seed-based artificial miRNA design was highly inefficient, as the majority of miRNAs with even perfect seed matches did not repress either target. Moreover, commonly used target prediction programs did not substantially discriminate effective artificial miRNAs from ineffective ones, indicating that current algorithms do not fully capture the features important for artificial miRNA targeting and are not yet sufficient for designing artificial miRNAs. Our analysis suggests that additional factors are strong determinants of the efficacy of miRNA-mediated target repression and remain to be discovered. 293T cells were transiently transfected with artificial miRNAs or non-targeting control (Allstars siRNA, Qiagen). Three replicate transfections were performed for each miRNA or control. Total RNA was extracted 48 hours after transfection.

2014-02-28 | E-GEOD-50249 | biostudies-arrayexpress

Global analysis of off-target genes by GAPDH siRNA

Project description:A GAPDH siRNA was overexpressed in HeLa cells and microarray expression profiling was performed to experimentally identify the off-targets of this siRNA at the transcriptome level. Among all predicted siRNA off-targets by MirTarget2, the majority were downregulated by at least 10% (i.e. less than 90% remaining gene expression). Moreover, more than one fifth were downregulated by at least 30%. The gene knockdown data is consistent with previous observations that siRNA off-targets are generally silenced at a more moderate level as compared to silencing of the single intended target GAPDH siRNA was transfected into HeLa cells, and the unintended off-targets was identified by microarrays 24 hours post transfection.

2013-10-03 | E-GEOD-41924 | biostudies-arrayexpress

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data