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Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells.

ABSTRACT: Gene targeting has two important applications. One is the inactivation of genes ("knockouts"), and the second is the correction of a mutated allele back to wild-type ("gene therapy"). Central to these processes is the efficient introduction of the targeting DNA into the cells of interest. In humans, this targeting is often accomplished through the use of recombinant adeno-associated virus (rAAV). rAAV is presumed to use a pathway of DNA double-strand break (DSB) repair termed homologous recombination (HR) to mediate correct targeting; however, the specifics of this mechanism remain unknown. In this work, we attempted to generate Ku70-null human somatic cells by using a rAAV-based gene knockout strategy. Ku70 is the heterodimeric partner of Ku86, and together they constitute an end-binding activity that is required for a pathway [nonhomologous end joining (NHEJ)] of DSB repair that is believed to compete with HR. Our data demonstrated that Ku70 is an essential gene in human somatic cells. More importantly, however, in Ku70(+/-) cells, the frequency of gene targeting was 5- to 10-fold higher than in wild-type cells. RNA interference and short-hairpinned RNA strategies to deplete Ku70 phenocopied these results in wild-type cells and greatly accentuated them in Ku70(+/-) cell lines. Thus, Ku70 protein levels significantly influenced the frequency of rAAV-mediated gene targeting in human somatic cells. Our data suggest that gene-targeting frequencies can be significantly improved in human cells by impairing the NHEJ pathway, and we propose that Ku70 depletion can be used to facilitate both knockout and gene therapy approaches.

SUBMITTER: Fattah FJ

PROVIDER: S-EPMC2438404 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells.

Fattah Farjana J FJ Lichter Natalie F NF Fattah Kazi R KR Oh Sehyun S Hendrickson Eric A EA

Proceedings of the National Academy of Sciences of the United States of America 20080618 25

Gene targeting has two important applications. One is the inactivation of genes ("knockouts"), and the second is the correction of a mutated allele back to wild-type ("gene therapy"). Central to these processes is the efficient introduction of the targeting DNA into the cells of interest. In humans, this targeting is often accomplished through the use of recombinant adeno-associated virus (rAAV). rAAV is presumed to use a pathway of DNA double-strand break (DSB) repair termed homologous recombin ...[more]

PMID: 18562296

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Project description:RNA editing by RNA specific adenosine deaminase acting on RNA (ADAR) is increasingly being found to alter microRNA (miRNA) regulation. Editing of miRNA transcripts can affect their processing, as well as which messenger RNAs (mRNAs) they target. Further, editing of target mRNAs can also affect their complementarity to miRNAs. Notably, ADAR editing is often increased in malignancy with the effect of these RNA changes being largely unclear. In addition, numerous reports have now identified an array of miRNAs that directly contribute to various malignancies although the majority of their targets remain largely undefined. Here we propose that modulating the targets of miRNAs via mRNA editing is a frequent occurrence in cancer and an underappreciated participant in pathology. In order to more accurately characterize the relationship between these two regulatory processes, this study examined RNA editing events within mRNA sequences of two breast cancer cell lines (MCF-7 and MDA-MB-231) and determined whether or not these edits could modulate miRNA associations. Computational analyses of RNA-Seq data from these two cell lines identified over 50,000 recurrent editing sites within human mRNAs, and many of these were located in 3' untranslated regions (UTRs). When these locations were screened against the list of currently-annotated miRNAs we discovered that editing caused a subset (~9%) to have significant alterations to mRNA complementarity. One miRNA in particular, miR-140-3p, is known to be misexpressed in many breast cancers, and we found that mRNA editing allowed this miRNA to directly target the apoptosis inducing gene DFFA in MCF-7, but not in MDA-MB-231 cells. As these two cell lines are known to have distinct characteristics in terms of morphology, invasiveness and physiological responses, we hypothesized that the differential RNA editing of DFFA in these two cell lines could contribute to their phenotypic differences. Indeed, we confirmed through western blotting that inhibiting miR-140-3p increases expression of the DFFA protein product in MCF-7, but not MDA-MB-231, and further that inhibition of miR-140-3p also increases cellular growth in MCF-7, but not MDA-MB-231. Broadly, these results suggest that the creation of miRNA targets may be an underappreciated function of ADAR and may help further elucidate the role of RNA editing in tumor pathogenicity.

Dataset Information

Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells.

Publications

Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells.

Similar Datasets

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