p53 Activation by Knockdown Technologies
By Mara E. Robu, Jon D. Larson, Aidas Nasevicius, S. Beiraghi, Charles Brenner, S. Farber, S. Ekker. PLoS Genetics, 2007
1,081 citations recorded by the source corpus.
Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies.
Subfields: Cancer Research, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/bc419b76e5a2d861012d39a0e6b513676e03a943
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Citations in the corpus
- MicroRNA-125b is a novel negative regulator of p53. (2009, background): “Exposure to H-7 leads to an increased import of p53 into the nucleus, where p53 becomes active and induces apoptosis (Ronca et al. 1997).”
- Controlling morpholino experiments: don't stop making antisense (2008, background): “The non-specific neural defects observed in the above experiments, and in 15-20% of all MO experiments carried out in zebrafish, are likely, as explained below, to derive from the activation of p53 and consequent p53-induced apoptosis (Robu et al., 2007).”
- Reverse genetic screening reveals poor correlation between Morpholino-induced and mutant phenotypes in zebrafish (2014, background): “While it is possible to alleviate off-target phenotypes by simultaneously reducing p53 levels (Robu et al., 2007), the mechanism of p53 activation is unknown.”