MicroRNA-like off-target transcript regulation by siRNAs
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ABSTRACT:
PROVIDER: PRJNA110373 | ENA |
REPOSITORIES: ENA
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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: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.
2009-02-01 | GSE14073 | GEO
Project description:Interventions: Gold Standard:colonoscopy and pathology;Index test:Stool multi-target DNA and microRNA-135b
Primary outcome(s): Stool multi-target DNA and microRNA-135b
Study Design: Diagnostic test for accuracy
| 2681097 | ecrin-mdr-crc
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
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: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:microRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis
2019-02-19 | PXD011882 | Pride
Project description:Despite their prominent role in transposon silencing, expression of endo-siRNAs is not limited to the “non-self” DNA elements. Transcripts of protein-coding genes (“self” DNA) in some cases also produce endo-siRNAs in yeast, plants, and animals [1]. How cells distinguish these two populations of siRNAs to prevent unwanted silencing of self-genes in animals is not well understood. To address this question, we examined the expression of ectopic siRNAs from an LTR retrotransposon in C. elegans germline. We found that the abundance of ectopic siRNAs was dependent on their homologous target genes: ectopic siRNAs against genes expressed only in somatic cells can be abundantly expressed. In contrast, ectopic siRNAs against germline-expressed genes are often suppressed. This phenomenon, which we termed “target-directed siRNA suppression”, is dependent on the target mRNA and requires germline P-granule components. We found that siRNA suppression can also occur to naturally produced endo-siRNAs. We suggest that siRNA suppression plays an important role in regulating siRNA expression and preventing self-genes from aberrant epigenetic silencing.
2022-02-19 | GSE196847 | GEO
Project description:Target-directed degradation (TDD) of microRNAs (miRNAs) plays an important role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of Drosophila cells have target sites resembling those that trigger miRNA TDD, raising the question as to whether they too might undergo such regulation. Here, we find that at least seven of these siRNAs are indeed sensitive to TDD when loaded into AGO1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into AGO1, these siRNAs are not detectably regulated by TDD because most molecules are loaded into AGO2, the Argonaute paralog that preferentially associates with siRNAs, and we find that siRNAs and miRNAs loaded into AGO2 are insensitive to TDD. One explanation for the protection of these small RNAs loaded into AGO2 is that these small RNAs are 2′-O-methylated at their 3′ termini. However, contrary to previous proposals, 2′-O-methylation does not protect these RNAs from TDD, which indicates that their protection is instead conferred by features of the AGO2 protein itself. Together, these observations clarify the requirements for regulation by TDD and revise our understanding of the role of 2′-O-methylation in small-RNA biology.
2021-01-05 | GSE163938 | GEO
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