Mechanistic insights into RNA binding and RNA-regulated RIG-I ubiquitination by TRIM25

Abstract

TRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25’s E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA-binding have been described, the precise RNA binding site has not been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. Mutational analysis shows that RNA binding affects ubiquitination of RIG-I in mammalian cells. In addition, we present a simple model system for RNA-induced liquid-liquid phase separation of TRIM25 in vitro, resembling previously observed cellular RNA granules, that facilitates the recruitment of RIG-I.

Validation and classification of RNA binding proteins identified by mRNA interactome capture

“…As opposed to TXN, scFPR1, and SNTB1, NMR resonances of TRIM25 exhibited clear CSPs upon addition of five molar equivalents of poly(U) 15-mer RNA, confirming its ability to bind single-stranded RNA (Figure 1D). Follow-up NMR studies and detailed biophysical characterization of the RNA binding of TRIM25 has been published in the meantime (Haubrich et al, 2020), where we could confirm that TRIM25 has a preference for stem loop RNA and binds single-stranded as well as structured RNA on two different binding sites on the PRYSPRY domain. Additionally, we could also confirm that the coiled-coil binds to RNA synergistically with the PRY/SPRY domain.…”

“…Additionally, we could also confirm that the coiled-coil binds to RNA synergistically with the PRY/SPRY domain. TRIM25 binding by RNA has an effect on RIG-I ubiquitination and the interferon response, suggesting an involvement of RNA in the host defense against viral infection (Haubrich et al, 2020).…”

“…Among the putative novel RBPs that exhibit direct RNA binding, some (Tm1-I/C, TRIM-NHL and TRIM-SPRY proteins) can be regarded as bona fide RBPs with their RNA binding ability confirmed both in isolation in vitro and in vivo (Haubrich et al, 2020). EB1, on the other hand exhibits RNA binding in vitro , but its biological significance could not be validated in vivo .…”

“…Moreover, about half of the identified proteins lacked classical RNA binding domains and many did not even have previously known functions related to RNA (Hentze et al, 2018). Subsequently, a few of these proteins were tested and validated to be bona fide RNA binding proteins, for example p62 or the TRIM family proteins Brain tumor and TRIM25 (Choudhury et al, 2017a; Haubrich et al, 2020; Horos et al, 2019; Loedige et al, 2015; Loedige et al, 2014). Of note, a verified RNA binding protein is the cytoskeletal protein APC (Adenomatous polyposis coli).…”

Viral Evasion of RIG-I-Like Receptor-Mediated Immunity through Dysregulation of Ubiquitination and ISGylation

“…Structural analysis highlighted that TRIM25 RING domain dimerization leads to higher-order oligomerization and catalytic activation of TRIM25, which is required for RIG-I ubiquitination and subsequent activation [ 35 ]. Recent studies have shown that TRIM25 also possesses RNA-binding activity, via its PRY/SPRY domain and coiled-coil domain (CCD), which regulates the ubiquitin E3 ligase activity of TRIM25, its oligomeric state, localization within the cell, and antiviral activity [ 36 , 37 , 38 ]. Several viruses have evolved to antagonize the TRIM25-RIG-I signaling axis, thereby limiting IFN responses ( Figure 1 ).…”