Close

AGO1x prevents dsRNA-induced interferon signaling to promote breast cancer cell proliferation

Souvik Ghosh, Joao C Guimaraes, Manuela Lanzafame, Alexander Schmidt, Afzal Pasha Syed, Beatrice Dimitriades, Anastasiya Börsch, Shreemoyee Ghosh, Ana Luisa Correia, Johannes Danner, Gunter Meister, Luigi M. Terracciano, Salvatore Piscuoglio, Mihaela Zavolan

Posted on: 5 May 2019 , updated on: 7 May 2019

Preprint posted on 10 April 2019

Article now published in The EMBO Journal at http://dx.doi.org/10.15252/embj.2019103922

When do stop signs become optional? Ghosh et al. report an important role for an AGO1 isoform generated by translational readthrough.

Selected by Lorenzo Lafranchi

 

Background

Programmed and efficient stop codon readthrough has been initially observed in viruses, but its occurrence has recently been confirmed in other organisms, including humans. Translational readthrough diversifies the proteome by producing low abundant protein isoforms carrying C-terminal extensions. Often, the function of these isoforms differs from that of the canonical protein. The efficiency of translational readthrough depends on a variety of factors. For example, cis-acting elements located in 3’ untranslated region (3’ UTR) of mRNAs can facilitate stop codon readthrough. In the case of the vascular endothelial growth factor A (VEGF-A), binding of the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 protein to the 3’ UTR of the mRNA promotes synthesis of an extended isoform dubbed VEGF-Ax (Eswarappa et al, 2014). The same study identified the mRNA of Argonaute 1 (AGO1), a protein well known for its role in RNA-mediated post-transcriptional gene silencing, as a putative target of translational readthrough. However, a thorough characterization of the arising AGO1 isoform was missing so far.

 

Key findings

Corroborating the hypothesis that the Argonaute 1 gene is prone to translational readthrough, the authors observed that the region downstream of the AGO1 stop codon is highly conserved across vertebrates. Western blot analysis of AGO1 expression levels in different cell lines revealed a second band of higher molecular weight in addition to the main, expected AGO1 signal. Interestingly, the upper band is more prominent in a breast cancer cell line, compared to the two other cancer cell lines used. Based on these data, the authors speculated that an amplification of the AGO1 locus in breast and ovarian cancer xenografts results in higher translational readthrough of the AGO1 gene. To confirm that the slower-migrating band belongs to a readthrough-dependent AGO1 isoform, the authors raised an antibody directed to the readthrough region of the peptide. With this tool in hand, and supporting their finding with mass spectrometry, the authors convincingly show that translational readthrough takes place on the AGO1 transcript, resulting in the AGO1x isoform.

 

Unlike canonical AGO1, which resides in the cytoplasm, AGO1x is enriched around the nucleoli, suggesting a different functional role for this isoform. To better understand AGO1x function, two independent mutant cell lines lacking the AGO1x isoform were generated directing the CRISPR/Cas9 system at the predicted readthrough region. Characterizing the mutants, the authors noticed that in absence of AGO1x cell growth and mobility are reduced. Consistently, immunohistochemistry of breast cancer tissues revealed that AGO1x is principally expressed in highly proliferative cells. To define how AGO1x is supporting cell proliferation, the authors analyzed the transcriptome of control and mutant cell lines. RNA sequencing data showed that hundreds of genes are differentially-expressed in AGO1x-deficient cells and these changes are consistent for the two mutant lines. A detailed analysis revealed that expression of genes related to the interferon alpha response and to the apoptosis pathways are increased in the mutant cells relative to control. In line with this observation, treatment with ruxolitinib, an inhibitor of the interferon response fully rescued the growth defect of the mutant cell lines.

 

By testing their hypothesis that activation of the interferon response in the AGO1x-deficient cells could be due to the accumulation of cytoplasmic double-stranded RNA (dsRNA), the authors demonstrate that the levels of several intracellular dsRNA sensors are increased in the mutant cells compared to the parental cell line. As suggested by the increased amount of dsRNA sensors, higher levels of dsRNA, in particular belonging to rRNAs and GC-rich mRNAs, were detected in the absence of AGO1x. Finally, the Polyribonucleotide nucleotidyltransferase 1 (PNPT1) and the ATP-dependent RNA helicase A (DHX9) were identified as interaction partners of AGO1x. Altogether, these data suggest a role for this newly-discovered complex in sequestering and, possibly, degrading dsRNA. This protective mechanism seems to be particularly important for highly proliferative cells, relying on an increased transcription of ribosomal DNA.

 

What I like about this work and future direction

It has recently been proposed that cancer cells rely on an alternative translational program, skewed towards oncogenic mRNA translation and sustained translation of upstream open reading frames (uORF). Similarly, the data presented in this paper suggest that translational readthrough could be an additional feature of translational reprogramming of cancer cells. Nevertheless, more data are required to understand if translational readthrough in cancer cells is specifically directed to a subset of genes, as in the case of Argonaute 1, or is a widespread phenomenon.

 

Questions

The authors speculate that amplification of the AGO1 locus in breast and ovarian cancers results in higher expression of AGO1x. Why would this amplification not result in a concomitant increase of the canonical gene product?

Is it possible that increased levels of the heterogeneousribonucleoprotein A2/B1 in cancer cells (or generally in proliferative cells) are responsible for the increment in translational readthrough? Did you compare hnRNP A2/B1 levels between the cell lines used in this study?

Does depletion of hnRNP A2/B1 recapitulate the phenotypes observed in the AGO1x-deficient cell lines?

Is it possible to use the MS data presented in fig. 1e to define which amino acid is used to suppress the stop codon of the AGO1 gene?

 

References

Eswarappa SM, Potdar AA, Koch WJ, Fan Y, Vasu K, Lindner D, Willard B, Graham LM, DiCorleto PE, Fox PL. Programmed translational readthrough generates antiangiogenic VEGF-Ax. Cell. 2014 Jun 19;157(7):1605-18.

 

doi: https://doi.org/10.1242/prelights.10500

Read preprint (No Ratings Yet)

Author's response

Joao Guimaraes and Mihaela Zavolan shared

  • The authors speculate that amplification of the AGO1 locus in breast and ovarian cancers results in higher expression of AGO1x. Why would this amplification not result in a concomitant increase of the canonical gene product?

This is an interesting and likely complex question that we have not yet investigated. Indeed, if the readthrough is not specifically facilitated in the cancer cells at the expense of the canonical product, the canonical product should increase as well. Furthermore, there are reports that Ago proteins influence each other’s expression, and that the localization of Ago proteins varies between cell types and conditions. Overall, it seems that the functions of Ago family proteins have still not been fully charted.

  • Is it possible that increased levels of the heterogeneousribonucleoprotein A2/B1 in cancer cells (or generally in proliferative cells) are responsible for the increment in translational readthrough? Did you compare hnRNP A2/B1 levels between the cell lines used in this study?

The AGO1 3’UTR is predicted to have a binding site for hnRNP A2/B1 and therefore this protein could regulate AGO1 stop codon readthrough, as it has been previously described for VEGF-Ax. In our study, we focused on the Ago1x function, about which nothing was known, rather than on the regulation of Ago1x expression. Clearly there is much more work to be done in this area, especially given the increased expression of Ago1x in the context of cancer cells. The mechanisms behind this expression pattern are an indeed what we would like to learn about in the immediate future.

  • Does depletion of hnRNP A2/B1 recapitulate the phenotypes observed in the AGO1x-deficient cell lines?

As said, we have not tested whether hnRNP A2/B1 is the factor behind Ago1x expression in cancer cells. A few recent studies showed that inhibition of HNRNPA2B1 has potential antitumor effects in a pancreatic cancer cell model (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223355/) and in glioblastoma (http://cancerres.aacrjournals.org/content/71/13/4464). Although these findings are consistent with the notion that HNRNPA2B1 regulates the translation readthrough of AGO1, this hypothesis needs to be directly tested.

  • Is it possible to use the MS data presented in fig. 1e to define which amino acid is used to suppress the stop codon of the AGO1 gene?

We did wonder about this, but unfortunately, the peptide that covers the stop codon region is too short to be resolved by mass spectrometry. One would have to experiment with different digestion methods during sample preparation so that a peptide of appropriate size is generated. Clearly, there is much more to be learned about this peculiar and highly conserved isoform, and we hope that our findings will prompt further studies.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cancer biology category:

Mitochondria-derived nuclear ATP surge protects against confinement-induced proliferation defects

Ritobrata Ghose, Fabio Pezzano, Savvas Kourtis, et al.

Selected by 16 May 2024

Teodora Piskova

Cell Biology

Spatial transcriptomics elucidates medulla niche supporting germinal center response in myasthenia gravis thymoma

Yoshiaki Yasumizu, Makoto Kinoshita, Martin Jinye Zhang, et al.

Selected by 27 March 2024

Jessica Chevallier

Immunology

Invasion of glioma cells through confined space requires membrane tension regulation and mechano-electrical coupling via Plexin-B2

Chrystian Junqueira Alves, Theodore Hannah, Sita Sadia, et al.

Selected by 13 February 2024

Jade Chan

Cancer Biology

Also in the cell biology category:

Mitochondria-derived nuclear ATP surge protects against confinement-induced proliferation defects

Ritobrata Ghose, Fabio Pezzano, Savvas Kourtis, et al.

Selected by 16 May 2024

Teodora Piskova

Cell Biology

Lipid-Based Transfection of Zebrafish Embryos: A Robust Protocol for Nucleic Acid Delivery

Aslihan Terzi, Tiger Liao, Adrian Jacobo

Selected by 09 May 2024

Roberto Rodríguez-Morales

Genetics

Fetal brain response to maternal inflammation requires microglia

Bridget Elaine LaMonica Ostrem, Nuria Dominguez Iturza, Jeffrey Stogsdill, et al.

Selected by 24 April 2024

Manuel Lessi

Neuroscience

preLists in the cancer biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Anticancer agents: Discovery and clinical use

Preprints that describe the discovery of anticancer agents and their clinical use. Includes both small molecules and macromolecules like biologics.

 



List by Zhang-He Goh

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

Also in the cell biology category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close