ABSTRACT
Active hypusine-modified initiation elongation factor 5A is critical for cell proliferation and differentiation, embryonic development, and innate immune response of macrophages to bacterial infection. Here, we demonstrate that both virus infection and double-stranded RNA viral mimic stimulation induce the hypusination of eIF5A. Furthermore, we show that activation of eIF5A is essential for the replication of several RNA viruses including influenza A virus, vesicular stomatitis virus, chikungunya virus, mayaro virus, una virus, zika virus, and punta toro virus. Finally, our data reveal that inhibition of eIF5A hypusination using the spermidine analog GC7 or siRNA-mediated downmodulation of eIF5A1 induce upregulation of endoplasmic reticulum stress marker proteins and trigger the transcriptional induction of interferon and interferon-stimulated genes, mechanisms that may explain the broad-spectrum antiviral activity of eIF5A inhibition.
Subject(s)
RNA Viruses , Virus Diseases , Zika Virus Infection , Zika Virus , Antiviral Agents , Humans , Interferons , RNA, Double-Stranded , Virus ReplicationABSTRACT
The cellular response to DNA damage is an intricate mechanism that involves the interplay among several pathways. In this study, we provide evidence of the roles of the polyadenylation factor cleavage stimulation factor 50 (CstF-50) and the ubiquitin (Ub) escort factor p97 as cofactors of BRCA1/BARD1 E3 Ub ligase, facilitating chromatin remodeling during the DNA damage response (DDR). CstF-50 and p97 formed complexes with BRCA1/BARD1, Ub, and some BRCA1/BARD1 substrates, such as RNA polymerase (RNAP) II and histones. Furthermore, CstF-50 and p97 had an additive effect on the activation of the ubiquitination of these BRCA1/BARD1 substrates during DDR. Importantly, as a result of these functional interactions, BRCA1/BARD1/CstF-50/p97 had a specific effect on the chromatin structure of genes that were differentially expressed. This study provides new insights into the roles of RNA processing, BRCA1/BARD1, the Ub pathway, and chromatin structure during DDR.
Subject(s)
Adenosine Triphosphatases/genetics , BRCA1 Protein/genetics , Chromatin Assembly and Disassembly , Cleavage Stimulation Factor/genetics , DNA Damage , DNA Repair , Nuclear Proteins/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Adenosine Triphosphatases/metabolism , BRCA1 Protein/metabolism , Cleavage Stimulation Factor/metabolism , DNA-Binding Proteins/metabolism , Histones/genetics , Histones/metabolism , Humans , Nuclear Proteins/metabolism , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitination , mRNA Cleavage and Polyadenylation Factors/metabolismABSTRACT
Following DNA damage, mRNA levels decrease, reflecting a coordinated interaction of the DNA repair, transcription and RNA processing machineries. In this study, we provide evidence that transcription and polyadenylation of mRNA precursors are both affected in vivo by UV treatment. We next show that the polyadenylation factor CstF, plays a direct role in the DNA damage response. Cells with reduced levels of CstF display decreased viability following UV treatment, reduced ability to ubiquitinate RNA polymerase II (RNAP II), and defects in repair of DNA damage. Furthermore, we show that CstF, RNAP II and BARD1 are all found at sites of repaired DNA. Our results indicate that CstF plays an active role in the response to DNA damage, providing a link between transcription-coupled RNA processing and DNA repair.
Subject(s)
Cleavage Stimulation Factor/physiology , DNA Repair , Polyadenylation , DNA Damage , HeLa Cells , Humans , RNA Polymerase II/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , Ultraviolet RaysABSTRACT
BRCA1-associated RING domain protein BARD1, along with its heterodimeric partner BRCA1, plays important roles in cellular response to DNA damage. Immediate cellular response to genotoxic stress is mediated by a family of phosphoinositide 3-kinase-related protein kinases, such as ataxia-telangiectasia mutated (ATM), ATM and Rad3-related, and DNA-dependent protein kinase. ATM-mediated phosphorylation of BRCA1 enhances the DNA damage checkpoint functions of BRCA1, but how BARD1 is regulated during DNA damage signaling has not been examined. Here, we report that BARD1 undergoes phosphorylation upon ionizing radiation or UV radiation and identify Thr(714) as the in vivo BARD1 phosphorylation site. Importantly, DNA damage functions of BARD1 (i.e., inhibition of pre-mRNA polyadenylation and degradation of RNA polymerase II) are abrogated in T714A and T734A mutants. Our findings suggest that phosphorylation of BARD1 is critical for the DNA damage functions of the BRCA1/BARD1 complex.
Subject(s)
BRCA1 Protein/metabolism , DNA Damage/physiology , RNA, Messenger/antagonists & inhibitors , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , BRCA1 Protein/antagonists & inhibitors , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Cell Line, Tumor , Conserved Sequence , Humans , Molecular Sequence Data , Osteosarcoma/genetics , Osteosarcoma/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transfection , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/radiation effects , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/radiation effectsABSTRACT
Mammalian cells exhibit a complex response to DNA damage. The tumor suppressor BRCA1 and associated protein BARD1 are thought to play an important role in this response, and our previous work demonstrated that this includes transient inhibition of the pre-mRNA 3' processing machinery. Here we provide evidence that this inhibition involves proteasomal degradation of a component necessary for processing, RNA polymerase II (RNAP II). We further show that RNAP IIO, the elongating form of the enzyme, is a specific in vitro target of the BRCA1/BARD1 ubiquitin ligase activity. Significantly, siRNA-mediated knockdown of BRCA1 and BARD1 resulted in stabilization of RNAP II after DNA damage. In addition, inhibition of 3' cleavage induced by DNA damage was reverted in extracts of BRCA1-, BARD1-, or BRCA1/BARD1-depleted cells. We also describe corresponding changes in the nuclear localization and/or accumulation of these factors following DNA damage. Our results support a model in which a BRCA1/BARD1-containing complex functions to initiate degradation of stalled RNAP IIO, inhibiting the coupled transcription-RNA processing machinery and facilitating repair.
