The siRNA seed region is divided into two functionally different domains in RNA interference in response to 2´-OMe modifications
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PROVIDER: PRJNA778247 | ENA |
REPOSITORIES: ENA
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Project description:To investigate effects of siRNA with 2'-O-methyl (2'-Ome) modifications on genome-wide off-targetat, we analyzed gene expression profile of HeLa cells transfected by their siRNAs.
2022-01-11 | GSE188314 | GEO
Project description:In RNA interference (RNAi), small interfering RNA (siRNA) functions to suppress the expression of its target mRNA with perfect sequence complementarity. In a mechanism different from above, siRNA also suppresses unintended mRNAs with partial sequence complementarities, mainly to the siRNA seed region (nucleotides 2-8). This mechanism is largely utilized by microRNAs (miRNAs) and results in siRNA-mediated off-target effects. Thus, the siRNA seed region is considered to be involved in both RNAi and off-target effects. In this study, we revealed that the impact of 2'-O-methyl (2'-OMe) modification is different according to the nucleotide positions. The 2'-OMe modifications of nucleotides 2-5 inhibited off-target effects without affecting on-target RNAi activities. In contrast, 2'-OMe modifications of nucleotides 6-8 increased both RNAi and off-target activities. The computational simulation revealed that the structural change induced by 2'-OMe modifications interrupts base pairing between siRNA and target/off-target mRNAs at nucleotides 2-5 but enhances at nucleotides 6-8. Thus, our results suggest that siRNA seed region consists of two functionally different domains in response to 2'-OMe modifications: nucleotides 2-5 are essential for avoiding off-target effects, and nucleotides 6-8 are involved in the enhancement of both RNAi and off-target activities.
| S-EPMC8771963 | biostudies-literature
Project description:Differential gene expression assessed in siTNF-OMe-P treated animals showed significant correlation between improved colon integrity and clinical parameters of colitis with reduced TLR activation, tissue regeneration and reduced pro-inflammatory cytokines, as compared to all treatment groups. This data shows the changes in gene expression profile associated with effective silencing of TNFa by a chemically-modified siRNA in the colon of colitic mice. We performed genome-wide gene expression profiling of 5% DSS-treated mice after intrarectal administration of a)siRNA against TNFa (siTNF), b)siRNA against TNFa modified with O-Methyl nucleotides (siTNF-OMe), c)siRNA against TNFa modified with O-Methyl-propanediol nucleotides (siTNF-OMe-P) and d) siControl (scrambled siRNA).
2011-09-09 | E-GEOD-31906 | biostudies-arrayexpress
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
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 (“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
2006-09-12 | GSE5814 | GEO
Project description:RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA 'specificity' design rules.
| S-EPMC4676691 | biostudies-literature
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
Project description:Gene expression profiling comparisons of Neuro2a transfected with either mKIAA1583 (Tmem132a) siRNA or control (non-specific) siRNA. Keywords: siRNA, gene expression
2008-04-09 | GSE5676 | GEO
Project description:Exogenous siRNAs are commonly used to regulate endogenous gene expression levels for gene function analysis, genotype-phenotype association studies and for gene therapy. Exogenous siRNAs can target mRNAs within the cytosol as well as nascent RNA transcripts within the nucleus, thus complicating siRNA targeting specificity. To highlight challenges in achieving siRNA target specificity, we targeted an overlapping gene set that we found associated with a familial form of multiple synostosis syndrome type 4 (SYSN4). In the affected family, we found that a previously unknown non-coding gene TOSPEAK/C8orf37AS1 was disrupted and the adjacent gene GDF6 was downregulated. Moreover, a conserved long-range enhancer for GDF6 was found located within TOSPEAK which in turn overlapped another gene which we named SMALLTALK/C8orf37. In fibroblast cell lines, SMALLTALK is transcribed at much higher levels in the opposite (convergent) direction to TOSPEAK. siRNA targeting of SMALLTALK resulted in post transcriptional gene silencing (PTGS/RNAi) of SMALLTALK that peaked at 72 h together with a rapid early increase in the level of both TOSPEAK and GDF6 that peaked and waned after 24 h. These findings indicated the following sequence of events: Firstly, the siRNA designed to target SMALLTALK mRNA for RNAi in the cytosol had also caused an early and transient transcriptional interference of SMALLTALK in the nucleus; Secondly, the resulting interference of SMALLTALK transcription increased the transcription of TOSPEAK; Thirdly, the increased transcription of TOSPEAK increased the transcription of GDF6. These findings have implications for the design and application of RNA and DNA targeting technologies including siRNA and CRISPR. For example, we used siRNA targeting of SMALLTALK to successfully restore GDF6 levels in the gene therapy of SYNS4 family fibroblasts in culture. To confidently apply gene targeting technologies, it is important to first determine the transcriptional interference effects of the targeting reagent and the targeted gene.
| S-EPMC8393430 | biostudies-literature
Project description:Reporter ion interference remains a limitation of isobaric tag-based sample multiplexing. Advances in instrumentation and data acquisition modes, such as the recently developed real-time database search (RTS), can reduce interference. However, interference persists as does the need to benchmark upstream sample preparation and data acquisition strategies. Here, we present an updated Triple yeast KnockOut (TKO) standard as well as corresponding upgrades to the TKO Viewing Tool (TVT2.5, http://tko.hms.harvard.edu/). Specifically, we expand the TKO standard to incorporate the TMTpro18-plex reagents (TKO18). We also construct a variant thereof which has been digested only with LysC (TKO18L). We compare proteome coverage and interference levels of TKO18 and TKO18L data that were acquired under different data acquisition modes and analyzed using TVT2.5. Our data illustrate that RTS reduces interference while improving proteome coverage and suggest that digesting with LysC alone only modestly reduces interference, albeit at the expense of proteome depth. Collectively, the two new TKO standards coupled with the updated TVT represent a convenient and versatile platform for assessing and developing methods to reduce interference in isobaric tag-based experiments.
2022-01-06 | PXD029458 | Pride