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TRIM25 interacts with and mono-ubiquitinates UPF1. (A) Western blot of TRIM25 inducible cells 02 transfected with V5-tagged UPF1 in the presence or absence of 1 μg/mL dox. Lysates were subjected to FLAG 03 IP. Data are representative of three independent experiments. (B-C) Western blot of TRIM25 inducible cells 04 transfected with (B) V5-UPF1 or (C) V5-UPF1 mutants (K281R, K592R) and HA-Ub in the presence of 1 μg/mL 05 dox. Lysates were subjected to V5 IP. Data are representative of three independent experiments. 06
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The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it...
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... first validated that TRIM25 interacts with UPF1. To do so, we transfected V5-tagged UPF1 into TRIM25 79 inducible cell lines, then induced for TRIM25-WT or -R54P expression with dox, and performed a FLAG IP to 80 pull down TRIM25. We found that UPF1 is robustly detected only when TRIM25 is induced (Fig. 4A), validating 81 the TRIM25-UPF1 interaction identified in our co-IP/MS. To test the hypothesis that TRIM25 ubiquitinates UPF1, ...
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... more robustly mono-ubiquitinated only in the presence of TRIM25-WT and not ligase-deficient TRIM25-R54P 85 (~50% more by ImageJ quantification, Fig. 4B), suggesting that TRIM25 mono-ubiquitinates UPF1. We then 86 identified putative ubiquitination sites by selecting residues that are both identified in a previously published 87 ubiquitinome 47 and predicted via UbPred to be ubiquitinated (Score > 0.70), 48 and mutated these sites to arginine 88 (K281R, K592R). Whereas ubiquitination ...
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... selecting residues that are both identified in a previously published 87 ubiquitinome 47 and predicted via UbPred to be ubiquitinated (Score > 0.70), 48 and mutated these sites to arginine 88 (K281R, K592R). Whereas ubiquitination is unchanged in UPF1 K281R, the introduction of K592R abolishes 89 UPF1 ubiquitination in the presence of TRIM25-WT (Fig. 4C). Together, these results validate our co-IP/MS 90 identification of UPF1 as a novel TRIM25 substrate. ...
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... and -R54P in line with our previous results (Fig. 4A, 5A, MW ~135 kDa), we did not identify NME1 (Fig. 5A, 03 MW 20-25 kDa). We also performed the reverse IP where we pulled down myc-tagged NME1, but were unable 04 to find any TRIM25 interacting with NME1 (Fig. 5B). We hypothesized that this lack of TRIM25-NME1 interaction 05 could be due to functional differences between ectopically ...
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... copyright holder for this preprint this version posted March 17, 2022. ; https://doi.org/10.1101/2022.03.17.484727 doi: bioRxiv preprint UPF1 (Fig. 4C) overlaps with a predicted acetylation site on the same residue, and neighbors a predicted 14 phosphorylation site at T595, potentially modulating these other post-translational modifications of UPF1. 54 ...
Citations
The CCCH-type zinc finger antiviral protein (ZAP) in humans, specifically isoforms ZAP-L and ZAP-S, is a crucial component of the cell’s intrinsic immune response. ZAP acts as a post-transcriptional RNA restriction factor, exhibiting its activity during infections caused by retroviruses and alphaviruses. Its function involves binding to CpG (cytosine-phosphate-guanine) dinucleotide sequences present in viral RNA, thereby directing it towards degradation. Since vertebrate cells have a suppressed frequency of CpG dinucleotides, ZAP is capable of distinguishing foreign genetic elements. The expression of ZAP leads to the reduction of viral replication and impedes the assembly of new virus particles. However, the specific mechanisms underlying these effects have yet to be fully understood. Several questions regarding ZAP’s mechanism of action remain unanswered, including the impact of CpG dinucleotide quantity on ZAP’s activity, whether this sequence is solely required for the binding between ZAP and viral RNA, and whether the recruitment of cofactors is dependent on cell type, among others. This review aims to integrate the findings from studies that elucidate ZAP’s antiviral role in various viral infections, discuss gaps that need to be filled through further studies, and shed light on new potential targets for therapeutic intervention.
Certain re-emerging alphaviruses, such as chikungunya virus (CHIKV), cause serious disease and widespread epidemics. To develop virus-specific therapies, it is critical to understand the determinants of alphavirus pathogenesis and virulence. One major determinant is viral evasion of the host interferon response, which upregulates antiviral effectors, including zinc finger antiviral protein (ZAP). Here, we demonstrated that Old World alphaviruses show differential sensitivity to endogenous ZAP in 293T cells: Ross River virus (RRV) and Sindbis virus (SINV) are more sensitive to ZAP than o’nyong’nyong virus (ONNV) and CHIKV. We hypothesized that the more ZAP-resistant alphaviruses evade ZAP binding to their RNA. However, we did not find a correlation between ZAP sensitivity and binding to alphavirus genomic RNA. Using a chimeric virus, we found the ZAP sensitivity determinant lies mainly within the alphavirus non-structural protein (nsP) gene region. Surprisingly, we also did not find a correlation between alphavirus ZAP sensitivity and binding to nsP RNA, suggesting ZAP targeting of specific regions in the nsP RNA. Since ZAP can preferentially bind CpG dinucleotides in viral RNA, we identified three 500-bp sequences in the nsP region where CpG content correlates with ZAP sensitivity. Interestingly, ZAP binding to one of these sequences in the nsP2 gene correlated to sensitivity, and we confirmed that this binding is CpG-dependent. Our results demonstrate a potential strategy of alphavirus virulence by localized CpG suppression to evade ZAP recognition.
Up-frameshift protein 1 (UPF1) plays the role of a vital controller for transcripts, ready to react in the event of an incorrect translation mechanism. It is well known as one of the key elements involved in mRNA decay pathways and participates in transcript and protein quality control in several different aspects. Firstly, UPF1 specifically degrades premature termination codon (PTC)-containing products in a nonsense-mediated mRNA decay (NMD)-coupled manner. Additionally, UPF1 can potentially act as an E3 ligase and degrade target proteins independently from mRNA decay pathways. Thus, UPF1 protects cells against the accumulation of misfolded polypeptides. However, this multitasking protein may still hide many of its functions and abilities. In this article, we summarize important discoveries in the context of UPF1, its involvement in various cellular pathways, as well as its structural importance and mutational changes related to the emergence of various pathologies and disease states. Even though the state of knowledge about this protein has significantly increased over the years, there are still many intriguing aspects that remain unresolved.
