Background
The RNA-induced transcriptional silencing (RITS) complex is a multi-subunit protein complex that functions in mediating short interfering (si)RNA-induced transcriptional gene silencing/epigenetic silencing. Argonaute-1 (Ago1) is the only known protein component of the human RITS machinery. Elucidation of the RITS complex is essential to understanding nuclear RNAi and for siRNA therapeutic development. In this study we investigate the RITS components in human cells using a HIV-1 infection model and antiviral siRNA that induce HIV-1 transcriptional/epigenetic silencing.
Methods
Immunoprecipitation (IP) studies were performed to pulldown Ago1 associated proteins using the HIV-1 epigenetic silencing model (HIV-infected HeLa-T4+ cells transfected with anti-HIV siRNA, siPromA, compared to Scrambled siRNA), followed by mass spectrometry (MS) in nuclear soluble fractions, for protein identification. Protein modelling was performed for Ago1 and candidate proteins, and Ago1-domain mutants were generated to investigate candidate protein binding, as well as candidate protein CRISPR knockout (KO) cell lines. Fixed/Live cell imaging was performed in CRISPR KO cell lines compared to wild-type HeLaT4+ cell lines.
Results
Ago1 IP and MS analysis identified an Ago1-HNRNPU association during siRNA-induced HIV transcriptional/epigenetic silencing. IP pulldowns of Ago1 with mutant domains indicated the Ago1-PAZ domain interacts with HNRNPU. CRISPR KO resulted in downregulation of ~70% of HNRNPU mRNA transcripts and ~60% of protein and resulted in increased HIV replication in the siPromA-treated KO cultures compared to wild-type cells. Moreover, live cell imaging showed dynamic cellular trafficking of siPromA-AlexaFluor647, Ago1-GFP and HNRNPU-mCherry, with siRNA colocalizing with HNRNPU in the cytoplasm before loading onto Ago1 and nuclear trafficking of all three to induce transcriptional/epigenetic silencing inside nucleus.
Conclusion
This study demonstrates an Ago1 and HNRNPU interaction and identifies HNRNPU as a potential human RITS complex component. This aligns with HNRNPU being a multifunctional RNA-binding protein, with roles in various cellular processes, including alternative splicing, RNA stability, and transcriptional regulation.