f 12  (Santa Cruz Biotechnology)

Journal: The Journal of Biological Chemistry

Article Title: Argonaute 1 contributes to the transcriptional silencing of HIV-1

doi: 10.1016/j.jbc.2025.110612

Figure Lengend Snippet: Argonaute 1 (Ago1) interacts with initiating and elongating RNAPII in cells . A, Ago1 is recruited to the chromatin fraction in an RNA-dependent manner. Cytoplasmic, nucleoplasmic, and chromatin-associated protein extracts were prepared from HeLa cells either untreated (NT) or treated with actinomycin D (ActD) at a 10 μg/ml final concentration to block transcription. Untreated cells were further either mock-treated or incubated with RNase A. Cytoplasmic, nucleoplasmic, and chromatin fractions were loaded on SDS-PAGE, and Ago1 and RNAPII (RPB1) were visualized using indicated antibodies. For each condition, three lanes are shown: NT (no ActD, no RNase), RNase A (no ActD, treated with RNase A), and ActD (ActD treated, no RNase A). GAPDH, EXOSC10, and histone H3 (H3 Tot) serve as markers for the cytoplasmic, nucleoplasmic, and chromatin fractions, respectively (n = 3 independent experiment, a representative image is shown). B, Ago1 interacts with endogenous RNAPII in nuclei of HeLa cells. Endogenous RNAPII was immunoprecipitated from nuclear extracts using an antibody targeting RPB1, the largest subunit of RNAPII. shAgo1-mediated Ago1 downregulation (6 days post shRNA transduction) served as a negative control for the interaction. GAPDH was used as additional negative and fractionation control. A representative image from n = 3 independent experiments is shown. C, reverse coimmunoprecipitation experiments were performed in HEK-293T cells either overexpressing, or not, Ago1-FLAG-HA, using an anti-FLAG antibody, in the absence (−) or presence (+) of RNase treatment. Ser5-phosphorylated (RNAPII-S5P) and Ser2-phosphorylated (RNAPII-S2P) forms of RNAPII serve as markers of the initiating and elongating transcriptional complexes, respectively. A representative result from n = 3 independent experiments is shown. HEK-293T, human embryonic kidney 293T cell line; RNAPII, RNA polymerase II.

Article Snippet: Membranes were blocked in Tris-buffered saline with 0.1% Tween-20 and 5% milk or 5% bovine serum albumin for 30 min at room temperature, followed by overnight incubation at 4 °C with the specified primary antibodies: anti-Ago1 4B8 (SAB4200084; Sigma), anti-Ago1 D84G10 (5053; Cell Signaling), anti-Ago2 C34C6 (2897; Cell Signaling), anti-Dicer A-2 (sc-136981; Santa Cruz), anti-Drosha D28B1 (3364; Cell Signaling), anti-RNA Pol II F-12 (sc-55492; Santa Cruz), anti-RNA Pol II phospho-Ser2 E1Z3G (13499; Cell Signaling), anti-RNA Pol II phospho-Ser5 (ab5131; Abcam), anti-GAPDH (sc-47724; Santa Cruz), and anti-α-Tubulin (T9026; Sigma–Aldrich).

Techniques: Concentration Assay, Blocking Assay, Incubation, SDS Page, Immunoprecipitation, shRNA, Transduction, Negative Control, Fractionation, Control

Journal: The Journal of Biological Chemistry

Article Title: Argonaute 1 contributes to the transcriptional silencing of HIV-1

doi: 10.1016/j.jbc.2025.110612

Figure Lengend Snippet: Model of the role of Argonaute 1 (Ago1) in transcriptional repression of the HIV-1 promoter. Ago1 is recruited to the HIV-1 promoter likely through RNA-dependent interactions, potentially involving LTR-driven nascent transcripts, possibly noncoding RNAs, but independently of miRNAs. Once associated with chromatin and RNA polymerase II (RNAPII), Ago1 may contribute to transcriptional repression either directly or by stabilizing a repressive chromatin environment at the promoter. This repressive function appears specific to Ago1 and does involve neither canonical miRNA pathway components nor the microprocessor or HUSH complexes. While mechanistically distinct from the RITS complex in Saccharomyces pombe , whose closest functional counterpart in human cells is the HUSH complex, this mechanism may reflect an evolutionary echo of RNA-guided transcriptional silencing strategies observed in lower eukaryotes. Ago1 may then serve as a scaffold for chromatin-modifying machineries, facilitating heterochromatinization of the viral promoter and contributing to the HIV-1 transcriptional silencing. RITS, RNA-induced transcriptional silencing.

Article Snippet: Membranes were blocked in Tris-buffered saline with 0.1% Tween-20 and 5% milk or 5% bovine serum albumin for 30 min at room temperature, followed by overnight incubation at 4 °C with the specified primary antibodies: anti-Ago1 4B8 (SAB4200084; Sigma), anti-Ago1 D84G10 (5053; Cell Signaling), anti-Ago2 C34C6 (2897; Cell Signaling), anti-Dicer A-2 (sc-136981; Santa Cruz), anti-Drosha D28B1 (3364; Cell Signaling), anti-RNA Pol II F-12 (sc-55492; Santa Cruz), anti-RNA Pol II phospho-Ser2 E1Z3G (13499; Cell Signaling), anti-RNA Pol II phospho-Ser5 (ab5131; Abcam), anti-GAPDH (sc-47724; Santa Cruz), and anti-α-Tubulin (T9026; Sigma–Aldrich).

Techniques: Functional Assay