Background:
RNA interference (RNAi) is integral to gene silencing, with transcriptional gene silencing (TGS) involving Argonaute 1 (Ago1) and promoter-targeted siRNA. Despite identification of nuclear Ago1-siRNA complexes in human cells, their trafficking dynamics, key Ago1 domains, and the impact on gene expression are not fully understood.
Methods:
Leica 3D Thunder live-cell imaging was used to track AlexaFluor647-tagged siRNAs and Ago1-GFP in HIV-1NL4.3-infected HeLa T4 cells. RNA sequencing (RNA-seq) identified differentially expressed genes (DEGs) under Mock, PromA, and Ago1-PromA conditions, with GO enrichment analysis. Pearson correlation evaluated gene expression profiles, and Ago1 truncation mutants assessed the role of specific domains in TGS.
Results:
Live-cell imaging revealed significant trafficking of Ago1-siPromA complexes to the perinuclear region (528.9 vesicles, p<0.0001), inner nuclear membrane (265 vesicles, p<0.0001), and nuclear cavity (264 vesicles, p<0.0001), compared to controls. A strong colocalization was observed between Ago1-GFP and siPromA-AF647 (mean PCC=0.2285, p<0.0001), unlike with scrambled siRNA (mean PCC=0.1625, p<0.0001). RNA-seq identified 1,349 DEGs (p<0.05, log2FC cutoffs), with significant upregulation of PPP1R15A, HMOX1, and SNAPC2, and downregulation of CHRM2 and HIST1H3G. GO analysis indicated downregulation of chromosomal segregation pathways and upregulation of RNA processing pathways in Ago1-siPromA cells. Additionally, HIV-1 gag transcripts were reduced by 70% (p<0.0001). Epigenetic analysis showed increased H3K9me3 (5-fold, p<0.001), H3K27me3 (1.5-fold, p<0.01), and Ago1 (3-fold, p<0.001) in siPromA-treated cells. Mutants lacking Ago1’s L1, PIWI, PAZ, or N-terminal regions showed significant increases in Gag mRNA (p<0.0001), confirming their role in TGS.
Conclusion:
The study demonstrates that specific Ago1 domains are essential for siRNA-induced epigenetic silencing of HIV. Significant alterations in gene expression and repressive epigenetic modifications underscore the potential of the Ago1-siPromA complex in the ‘Block and Lock’ strategy for achieving a functional HIV cure.