TDRD3: The Important Role in Promoting IFN Signaling by G3BP1

Methylation and demethylation of the RGG domain of G3BP1 drive stress granule (SG) dynamics; methylation is driven by protein arginine methyltransferases (PRMTs) 1 and 5. asymmetric methylation via PRMT1 is a major driver of the SG-building process and symmetric methylation via PRMT5 might regulate an unknown function in SG biology.

Tudor domain protein 3 (TDRD3) is one such effector molecule that "reads" methylarginine marks on histones and the C-terminal region of RNAPII. Crucially, genome-wide TDRD3 is very tightly linked to gene promoters and it appears to be in promoter-proximal R-loops. The fact that TDRD3 - a methyl-reader protein - is localized to SGs is due in part to the potential for methylation of G3BP1 and may recruit innate immune factors to SGs. Moreover, TDRD3 can, when overexpressed, also create SG-like structures independently of G3BP1. TDRD3 and G3BP1 are missing and the viral replication is promoted; TDRD3 is cleaved by the enteroviral protease 2A in the presence of the virus. If cells are deficient in TDRD3 and G3BP1, not all IFN effectors are transactivated to dsRNA. TDRD3 is regulated in different ways by G3BP1. TDRD3 could be a novel and critical regulator of host antiviral activity.

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Tudor Domain Protein 3 Physiological Activities

TDRD3 is a protein that assists G3BP1 in cell-mediated IFN signaling. IFN signalling is an aspect of the immune system that can modulate both antiviral and anti-tumor immunity. Another protein is G3BP1 which communicates with TDRD3 to influence the IFN signals. We can learn more about the regulation of immune responses and generate novel concepts for treatment and prevention of related conditions if we can figure out what role TDRD3 plays in this. The physiology of TDRD3 is the following:

Tudor Domain Protein 3 Induces SG-like Lesions

TDRD3 overexpression generates G3BP1- and Tia1-positive granules in cells that resemble most highly SGs produced during stress or G3BP1 overexpression. TDRD3-GFP was overexpressed in cells that did not have either G3BP1 or G3BP2 and granules do form in this cell. TDRD3-triggered granules also co-opt poly-A binding protein (PABP) and translation activators eIF3B, eIF4E and eIF4G1. If there are TDRD3-positive foci within the same G3BP-deficient cells. Loss of oxidative stress is critical for damage to SG after arsenite stress, when we apply ARS in G3BP-deficient cells. No one need be G3BP1 or G3BP2 in order for TDRD3 to condensation into SGs and, for TDRD3-driven SG-like foci, recruitment of canonical stress granule components as measured did not depend on G3BP1 or G3BP2, and TDRD3 overexpression also shut down host translation.

Fig. 1 TDRD3 is recruited to SGs and G3BP1 mutants (Deater, M., et al. 2022).

Work Together to Restrict Enterovirus Replication

In cells lacking G3BP1, viral replication rose slightly after 24 h but only at 24 h; TDRD3-KD cells seemed to inhibit viral replication just a bit. But absent or reduced G3BP1 and TDRD3, viral replication increased after 48 or 72 h to greater heights, and Coxsackievirus B3 (CVB3) was more infectious in these missing/reduced cells. This was also verified with the determination of TD50 at 50%. The same but weaker effects were seen with control virus (VSV) and Bengal virus. That means G3BP1 and TDRD3 co-act to produce antiviral effects, with a major impact on viral load.

TDRD3 seems to be an important actor in promoting IFN-I effector attraction to SGs and is unknown to be involved in SG assembly, which makes it a prime candidate for viral hijacking. 2A pro blocks SGs and reduces IFN- transcription, but no SG nucleation elements were cleaved by 2A pro. TDRD3 affects recruitment of innate immune signalling effectors to SGs and transcriptional activation of most ISGs of the interferon signalling system. TDRD3 and G3BP1 were both knocked out and enteroviral growth was improved by orders of magnitude when they were knocked out together, according to the quantification of viral genomes and infectivity studies. Additionally, when the infection level was very high MOI, enteroviral replication and host translational shutdown were not dissimilar, which implied some functional integration of TDRD3 and G3BP1 at the viral level.

Modulate Transcriptional Activation of Biological Factors in Tandem

Other interesting sequences of IFN-type transcriptional activation when G3BP1 or TDRD3 are missing, or both. More important here is that we see that G3BP1 induces transcriptional IFN- (type 1 IFN signaling) while TDRD3 induces transcription of IFN-1 (type 3 IFN signaling). In light of these results, it might be that G3BP1 is more involved in the regulation of type 1 IFN signaling while TDRD3 is more involved in controlling the expression of IFN-1. TDRD3 and G3BP1, either individually or together, seem to control the expression of the JAK-STAT signaling suppressors SOCS1 and SOCS3, with G3BP1 more prominently regulating the signalling of SOCS3.

Conclusion

Without the core nucleator G3BP1, TDRD3 can accumulate stress granule (SG)-like particles, and those particles can incubate signaling effectors. We also identified connections between TDRD3 and type 3 IFN, inflammation, and G3BP1 and type 1 IFN expression. G3BP1 and TDRD3 could cooperate to power the IFN response. This new role of TDRD3 as a particle 'mainstay' and potential antiviral protein, and the idea that it might act in concert with G3BP1, help us to see its role in the cytoplasm.

TDRD3 could recruit these factors because TDRD3 is a new antiviral protein that is cut up by the enterovirus 2A protease. This knockdown of G3BP1 and TDRD3 in cells led to differential transcriptional regulation of many IFN effectors, and only G3BP1 and TDRD3 could make such a complex pattern.

References

1. Deater, M., et al. TDRD3 is an antiviral restriction factor that promotes IFN signaling with G3BP1. PLoS Pathogens. 2022, 18(1): e1010249.
2. Yuan, W., et al. TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution. Nucleic acids research. 2021, 49(15): 8573-8591.