p53 Activation by Knockdown Technologies

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Abstract
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. Recent advances in sequence-based approaches to “knockdown” gene function have opened the door to an array of approaches to uncover functions for genes of interest. Vertebrate knockdown strategies—such as morpholinos (MOs) in zebrafish or RNA interference-based strategies in mammalian systems—have been demonstrated to be effective, rapid, and cost-efficient reverse-genetic approaches for studying gene function. However, their deployment has to date been limited by a number of technical (genomic, biological, and off-targeting) hurdles. One of the notable and unexpected findings from our work using MOs has been a series of observations surrounding unanticipated effects that are independent of the intended gene target. We have identified and characterized a recently described p53 induction pathway due to off-targeting that appears to be shared between knockdown technologies. This study reconciles a series of unexpected findings that show p53 upregulation at the transcriptional level in a subset of short inhibitory RNA- and MO-treated vertebrate systems. Moreover, concurrent p53 knockdown provides a new approach to facilitate the identification of previously hidden gene functions. This study provides both a new gene knockdown enhancement tool as well as additional insight into an important and conserved pathway implicated in cellular toxicity.