Loss of Lamin A leads to the nuclear translocation of AGO2 and compromised RNA interference

Abstract

In mammals, RNA interference (RNAi) is mostly studied as a cytoplasmic event, however, numerous reports convincingly show nuclear localization of the AGO proteins. Nevertheless, the mechanism of nuclear entry remains to be fully elucidated, and the extent of nuclear RNAi further explored. We found that reduced Lamin A levels significantly induced nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. The translocation of AGO2 was accompanied by aggravated cell proliferation and we further found that the loss of Lamin A leads to EGFR and Src kinase activation, which regulates the turnover and stability of cytoplasmic AGO2. Furthermore, Lamin A KO significantly reduced the activity of nuclear RNAi. This was evident by AGO fPAR-CLIP in WT and Lamin A KO cells, where we observed ca 60% less efficiency of RNAi. Mass spectrometry of AGO interactome, from the nuclear fraction, indicated that AGO2 is in complex with FAM120A, a known interactor of AGO2 that reduces the activity of RNAi by competing with AGO2 transcript binding. Therefore, loss of Lamin A starts a signaling cascade that mediates nuclear AGO2 translocation to rapidly inhibit RNAi in order to facilitate cancer proliferation.

Nuclear RNAi Modulates Influenza A Virus Infectivity By Downregulating Type-I Interferon Response

“…This adds to the multiple proviral roles of NS1 56 . A limited number of previous reports already identified nuclear presence of AGO2 upon IAV infection 22,57 , however both utilized cell lines (A549 and HEK293T) which are already nuclear-AGO positive at steady state 25 , thus complicating the interpretation of the results. Herein, by using HEK293 cells, which are nuclear AGO negative at steady state, we could better mimic what happens in mouse lungs.…”

“…AGO2 may localize both in the cytoplasm and the nucleus of human cells 25 . For instance, we previously demonstrated the absence of nuclear AGO2 in HEK293 cells 21,25 However, we observed a high degree of nuclear AGO2 in HEK293T cells, which are HEK293 cells transformed with SV40 large T (LT) antigen, the master regulator of polyomaviruses 26 To address this, we took advantage of PR8 strain of IAV as a model system. HEK293 cells were infected with PR8-IAV at several multiplicity of infection (MOI) 10 for 16 IAV consists of eight gene segments that encode up to 17 viral proteins 28 .…”

“…Samples were cleared with centrifugation at 12 000 g for 20 minutes and the supernatant collected. Biochemical fractionation assay was done as previously described 25 . Briefly, cell pellets were gently dissolved in a hypotonic lysis buffer (10 mM Tris-HCl, pH 7.6, 10 mM NaCl, 3 mM MgCl2, 0.3% NP-40, 10% Glycerol), supplemented with a protease inhibitor cocktail, with gentle pipetting up and down and collected with centrifugation for 2 minutes at 200 g. The supernatant was cleared with centrifugation at 12 000 g for 20 minutes and the supernatant stored as the cytoplasmic fraction.…”

Nuclear localization of Argonaute 2 is affected by cell density and may relieve repression by microRNAs

“…Improved purification protocols that exclude endoplasmic reticulum from nuclear lysate, however, demonstrated that AGO protein and miRNAs were present in cell nuclei ( 10 , 11 , 13 , 16 , 34 , 42 , 44 , 47 , 63 , 78 , 93 ). miRNAs were demonstrated to load in cell cytoplasm prior to transport into the nucleus.…”

Biochemical Separation of Cytoplasmic and Nuclear Fraction for Downstream Molecular Analysis

“…For example, transcription factors such as p53 or NF-Kβ are mainly located in the cytoplasm, but upon receiving an activation signal they translocate into the nucleus and interact with specific DNA loci to mediate the transcription of target genes (Liu et al, 2018). Another protein, Argonaute 2 (AGO2), the main component of RNA interference pathway is usually found within the cytoplasm, yet, in some conditions, such as cell confluence or certain cancers, can relocate to the nucleus (Huang et al, 2024;Johnson et al, 2023;Lobo et al, 2024;Sarshad et al, 2018). Therefore, an appropriate protocol to identify and separate the subcellular protein localization is critical to understand the physiological function of shuttling and localized proteins.…”