IADR Abstract Archives
Differential effects of H/ACA RNA-binding proteins in cell proliferation and response to DNA damage
Objectives: H/ACA-ribonucleoproteins (RNPs) are comprised of four highly conserved, nucleolar proteins, dyskerin, NHP2, NOP10 and GAR1, and a function-specifying, non-coding H/ACA RNA. Loss of dyskerin function arrests cell proliferation and may attenuate DNA-damage responses. Since the four proteins are presumed obligate binding partners, we hypothesized that loss of NHP2, NOP10 and GAR1 function would mimic the cellular effects of dyskerin depletion.
Methods: Cell lines were human keratinocytes (OKF6-TERT2-oral and HaCaT-skin) and U2OS-osteosarcoma cells. ON-TARGETplus SMARTPool siRNAs were obtained from Dharmacon (Lafayette, CO). All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), and antibodies from Proteintech (Chicago, IL) or Santa Cruz (Santa Cruz, CA). Protein extraction, immunoblotting, indirect immunofluorescence, RNA extraction, and cell cycle analyses were performed using standard techniques. Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Invitrogen, Carlsbad, CA) was used for proliferation.
Results: While all four proteins were upregulated following ultraviolet-irradiation, GAR1 levels increased more rapidly. In contrast, only GAR1 expression increased in response to various chemical genotoxic agents, and showed an altered subcellular localization with a shift to the nucleoplasm after irradiation and doxorubicin treatments. SiRNA-mediated depletion of dyskerin, NHP2 or NOP10, respectively, destabilized the other two components and reduced H/ACA RNA levels, without appreciable effect on GAR1. In contrast, loss of GAR1 enhanced stability of the other proteins but had no effect on the RNAs. Yet, only loss of dyskerin and GAR1 arrested proliferation. In contrast, loss of function of each protein attenuated DNA-damage signaling, as measured by reduced accumulation of several DNA-damage biomarkers following genotoxic stress.
Conclusions: H/ACA-RNP core proteins have differential effects on cell proliferation and may exert a novel role in modulation of DNA-damage response signaling. Since the specific contribution of each protein was not equivalent, this suggests the possibility that at least some of the proteins, most notably GAR1, may potentially function independently of their respective roles within H/ACA-RNP complexes.
Methods: Cell lines were human keratinocytes (OKF6-TERT2-oral and HaCaT-skin) and U2OS-osteosarcoma cells. ON-TARGETplus SMARTPool siRNAs were obtained from Dharmacon (Lafayette, CO). All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), and antibodies from Proteintech (Chicago, IL) or Santa Cruz (Santa Cruz, CA). Protein extraction, immunoblotting, indirect immunofluorescence, RNA extraction, and cell cycle analyses were performed using standard techniques. Click-iT® EdU Alexa Fluor® 488 Imaging Kit (Invitrogen, Carlsbad, CA) was used for proliferation.
Results: While all four proteins were upregulated following ultraviolet-irradiation, GAR1 levels increased more rapidly. In contrast, only GAR1 expression increased in response to various chemical genotoxic agents, and showed an altered subcellular localization with a shift to the nucleoplasm after irradiation and doxorubicin treatments. SiRNA-mediated depletion of dyskerin, NHP2 or NOP10, respectively, destabilized the other two components and reduced H/ACA RNA levels, without appreciable effect on GAR1. In contrast, loss of GAR1 enhanced stability of the other proteins but had no effect on the RNAs. Yet, only loss of dyskerin and GAR1 arrested proliferation. In contrast, loss of function of each protein attenuated DNA-damage signaling, as measured by reduced accumulation of several DNA-damage biomarkers following genotoxic stress.
Conclusions: H/ACA-RNP core proteins have differential effects on cell proliferation and may exert a novel role in modulation of DNA-damage response signaling. Since the specific contribution of each protein was not equivalent, this suggests the possibility that at least some of the proteins, most notably GAR1, may potentially function independently of their respective roles within H/ACA-RNP complexes.