title: PAF53 is essential in mammalian cells: CRISPR/Cas9 fails to eliminate PAF53 expression.
abstract: When mammalian cells are nutrient and/or growth factor deprived, exposed to inhibitors of protein synthesis, stressed by heat shock or grown to confluence, rDNA transcription is essentially shut off. Various mechanisms are available to accomplish this downshift in ribosome biogenesis. Muramatsu's laboratory (Hanada et al., 1996) first demonstrated that mammalian PAF53 was essential for specific rDNA transcription and that PAF53 levels were regulated in response to growth factors. While S. cerevisae A49, the homologue of vertebrate PAF53, is not essential for viability (Liljelund et al., 1992), deletion of yA49 results in colonies that grow at 6% of the wild type rate at 25°C. Experiments described by Wang et al. (2015) identified PAF53 as a gene "essential for optimal proliferation". However, they did not discriminate genes essential for viability. Hence, in order to resolve this question, we designed a series of experiments to determine if PAF53 was essential for cell survival. We set out to delete the gene product from mammalian cells using CRISPR/CAS9 technology. Human 293 cells were transfected with lentiCRISPR v2 carrying genes for various sgRNA that targeted PAF53. In some experiments, the cells were cotransfected in parallel with plasmids encoding FLAG-tagged mouse PAF53. After treating the transfected cells with puromycin (to select for the lentiCRISPR backbone), cells were cloned and analyzed by western blots for PAF53 expression. Genomic DNA was amplified across the "CRISPRd" exon, cloned and sequenced to identify mutated PAF53 genes. We obtained cell lines in which the endogenous PAF53 gene was "knocked out" only when we rescued with FLAG-PAF53. DNA sequencing demonstrated that in the absence of ectopic PAF53 expression, cells demonstrated unique means of surviving; including recombination or the utilization of alternative reading frames. We never observed a clone in which one PAF53 gene is expressed, unless there was also ectopic expression In the absence of ectopic gene expression, the gene products of both endogenous genes were expressed, irrespective of whether they were partially mutant proteins or not.
authors.affiliation: Depoartment of Cell Biology, The University of Oklahoma College of Medicine, Oklahoma City, OK 73104, United States. Electronic address: Lawrence-rothblum@ouhsc.edu.
authors.forename: Lawrence I
authors.initials: LI
authors.lastname: Rothblum
authors.affiliation: Depoartment of Cell Biology, The University of Oklahoma College of Medicine, Oklahoma City, OK 73104, United States.
authors.forename: Katrina
authors.initials: K
authors.lastname: Rothblum
authors.affiliation: Depoartment of Cell Biology, The University of Oklahoma College of Medicine, Oklahoma City, OK 73104, United States.
authors.forename: Eugenie
authors.initials: E
authors.lastname: Chang
mesh_terms: D000818:Animals; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
chemical_list: C484388:Paf53 protein, mouse; D014157:Transcription Factors
keywords: A49; CRISPR/Cas9; Nucleolus; PAF53; RNA polymerase I; Ribosomal RNA; Transcription; rDNA