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

ABSTRACT: In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event, however, in the last decade growing number of reports convincingly show nuclear localization of the AGO proteins. Nevertheless, the mechanism and the extent of nuclear RNAi remains to be fully elucidated. 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. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 evident by changes in cell morphology, increased cell proliferation and oncogenic miRNA expression. Furthermore, in SHSY5Y cells, AGO fPAR-CLIP in Lamin A KO cells revealed significantly reduced activity of RNAi. Further exploration of the nuclear AGO interactome by mass spectrometry indicated that AGO2 is in complex with FAM120A, an RNA binding protein and known interactor of AGO2. By performing FAM120A fPAR-CLIP we discovered that FAM120A co-binds AGO targets and that this competition reduces the activity of RNAi. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, RNAi impairment and selective upregulation of oncogenic miRNAs to facilitate cancer cell proliferation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE261593 | GEO | 2024/03/21

REPOSITORIES: GEO

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

Project description:In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event, however, in the last decade growing number of reports convincingly show nuclear localization of the AGO proteins. Nevertheless, the mechanism and the extent of nuclear RNAi remains to be fully elucidated. 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. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 evident by changes in cell morphology, increased cell proliferation and oncogenic miRNA expression. Furthermore, in SHSY5Y cells, AGO fPAR-CLIP in Lamin A KO cells revealed significantly reduced activity of RNAi. Further exploration of the nuclear AGO interactome by mass spectrometry indicated that AGO2 is in complex with FAM120A, an RNA binding protein and known interactor of AGO2. By performing FAM120A fPAR-CLIP we discovered that FAM120A co-binds AGO targets and that this competition reduces the activity of RNAi. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, RNAi impairment and selective upregulation of oncogenic miRNAs to facilitate cancer cell proliferation.

2024-03-21 | GSE261422 | GEO

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

Project description: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

2024-10-17 | PXD042899 | Pride

Loss of Lamin A leads to nuclear translocation of AGO2 mediated by EGFR signaling and inactivation of RNAi

Project description:In mammals, RNA interference (RNAi) is mostly studied as a cytoplasmic event, however, numerous reports convincingly showed nuclear localization of AGO proteins. Nevertheless, the mechanism of the nuclear entry remains to be fully elucidated, and the extent of nuclear RNAi explored. The nuclear import of large proteins requires active transport through the nuclear envelope (NE). Therefore, we hypothesized that the NE, and its composition, is important for nuclear AGO2 translocation. Lamin A knockout significantly induced nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cells. We unravelled that the loss of Lamin A leads to EGFR and Src kinase activation, which regulates the turnover and stability of cytoplasmic specifically AGO2. Furthermore, EGFR mediated phosphorylation at Y393 inhibits the activity of nuclear RNAi. This was evident by AGO fPAR-CLIP in WT and KO cells, where we observed a decrease in RNAi potential. Mass spectrometry of AGO interactome, from the nuclear fraction, indicated that AGO2 is in complex with FAM120A, which may further reduced the activity of RNAi by competing with AGO2 transcript binding. Therefore, loss of Lamin A starts a signalling cascade that mediates nuclear AGO2 translocation to rapidly inhibit RNAi.

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Sequestration of microRNA-mediated target repression by the Ago2-associated RNA binding protein FAM120A

Project description:Argonaute (Ago) proteins interact with various binding partners and play a pivotal role in microRNA (miRNA)-mediated silencing pathways. By utilizing immunoprecipitation followed by mass spectrometry to determine cytoplasmic Ago2 protein complexes in mouse embryonic stem cells (mESCs), we identified a putative RNA-binding protein FAM120A (also known as OSSA/C9ORF10) as an Ago2 interacting protein. Individual nucleotide resolution Cross-Linking and ImmunoPrecipitation (iCLIP) analysis revealed that FAM120A binds to homopolymeric tracts in 3´ UTRs of about 2,000 mRNAs, particularly poly(G) sequences. Comparison of FAM120A iCLIP and Ago2 iCLIP reveals that greater than one-third of mRNAs bound by Ago2 in mESCs are co-bound by FAM120A. Furthermore, such FAM120A-bound Ago2 target genes are not subject to Ago2-mediated target degradation. Reporter assays suggest that the 3´ UTRs of several FAM120A-bound miRNA target genes are less sensitive to Ago2-mediated target repression than those of FAM120A-unbound miRNA targets and FAM120A modulates them via its G-rich target sites. These findings suggest that Ago2 may exist in multiple protein complexes with varying degrees of functionality.

2019-07-09 | GSE128264 | GEO

Integrative transcriptomic and proteomic profiling of the effects of cell confluency on gene expression

Project description:In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in HEK293, A375, and SHSY5Y cell lines. As a consequence of cell confluency, AGO2 protein translocates into the nucleus. Therefore, we generated transcriptomic data using RNA sequencing to compare gene expression in subconfluent versus confluent cells, which highlighted significant alterations in gene regulation patterns directly corresponding to changes in cell density. Our study also encompasses miRNA profiling data obtained through small RNA sequencing, revealing miRNA expressional changes dependent on cellular confluency, as well as cellular localization. Finally, we derived proteomic data from mass spectrometry analyses following AGO1-4 immunoprecipitation, providing a comprehensive view of AGO interactome in both nuclear and cytoplasmic compartments under varying confluency. These datasets offer a detailed exploration of the cellular and molecular dynamics, influenced by cell confluency, presenting a valuable resource for further research in cellular biology, particularly in understanding the basic mechanisms of cell density in cancer cells.

2024-04-18 | PXD047707 | Pride

Integrative transcriptomic and proteomic profiling of the effects of cell confluency on gene expression

2024-04-19 | GSE249290 | GEO

Integrative transcriptomic and proteomic profiling of subconfluent and confluent cancer cells

Project description:In this study we examine the impact of cell confluency on gene expression. We focused on Argonaute (AGO) protein dynamics and associated gene and protein expression in HEK293, A375, and SHSY5Y cell lines. As a measure of cell confluency, we observed a profound translocation of AGO2 into the nucleus. Therefore, we generated transcriptomic data using RNA sequencing to compare gene expression in subconfluent versus confluent cells, which highlighted significant alterations in gene regulation patterns directly corresponding to changes in cell density. Our study also encompasses miRNA profiling data obtained through small RNA sequencing, revealing miRNA expressional changes dependent on cellular confluency, as well as cellular localization. Finally, we derived proteomic data from mass spectrometry analyses following AGO1-4 immunoprecipitation, providing a comprehensive view of AGO interactome in both nuclear and cytoplasmic compartments under varying confluency. These datasets offer a detailed exploration of the cellular and molecular dynamics, influenced by cell confluency, presenting a valuable resource for further research in cellular biology, particularly in understanding the basic mechanisms of cell density in cancer cells.

2024-04-19 | GSE255251 | GEO

Sites for Argonaute Binding within Human Nuclear RNA and their Impact on Alternative Splicing

Project description:Mammalian RNA interference (RNAi) is often linked to regulation in the cytoplasm. While synthetic RNAs regulate transcription and splicing, endogenous functions for nuclear RNAi have been obscure. Using enhanced crosslinking immunoprecipitation (eCLIP), we mapped AGO2 binding sites within nuclear RNA. The strongest AGO binding sites mapped to miRNAs. The prevalence of individual miRNAs was similar in the cytoplasm and the nucleus. Most statistically-significant AGO2 binding was within intron boundaries. RNAseq comparing AGO1, AGO2, AGO1/2, and AGO1/2/3 knockout to wild-type cells revealed that binding was associated with both up- and down-regulation of gene expression. We observed changes of splicing at genes with significant AGO binding at exon/intron junctions or within introns. Splicing changes were confirmed by RT-PCR and could be recapitulated by synthetic duplex RNAs that target the sites of AGO binding. Inhibition of miRNA action by addition of an anti-miR reversed miRNA-mediated control of splicing. We conclude: 1) That AGO binding sites occur throughout nuclear RNA but are often within introns; 2) The distribution of miRNAs is similar in the nucleus and cytoplasm; and 3) miRNAs can control gene splicing. Nuclear RNAi has the potential to be a natural regulatory mechanism but careful study will be necessary to identify critical RNA drivers of normal physiology and disease.

2021-02-10 | GSE161559 | GEO

Argonaute-CLIP delineates versatile, functional RNAi networks in Aedes aegypti, a major vector of human viruses

Project description:Argonaute (AGO) proteins bind small RNAs to silence complementary RNA transcripts and are central to RNA interference (RNAi). AGO-crosslinking immunoprecipitation (AGO-CLIP) has illuminated RNAi networks, but bioinformatic analysis is laborious and lack of experimental tools hinders its application outside of model organisms. RNAi is critical for regulation of gene expression and defense against viral infection in the Aedes aegypti mosquito, which transmits Zika, chikungunya, dengue, and yellow fever viruses to cause human disease. We developed AGO-CLIP for both mosquito AGO proteins and a universal, streamlined software package for CLIP analysis, identifying 230 novel small RNAs and 5,447 small RNA targets that comprise a comprehensive RNAi network map. We used this unique resource to predict repression of small RNA targets in specific mosquito tissues. Notably, this resource revealed unexpected AGO target preferences and uncovered a new mode of AGO-mediated repression, findings that have broad implications for the study of antiviral RNAi.

2020-09-04 | GSE157168 | GEO

The Core RNAi Machinery is Conserved Between the Cytoplasm and Nuclei of Human Cells

Project description:Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. Contrary to dogma, AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with FLAG-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that the core RNAi machinery is conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners.

2016-02-11 | MSV000079508 | MassIVE

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