Periodic Reporting for period 1 - UncoveRNAi (Deciphering the mechanisms of antiviral RNA interference in mammals)
Periodo di rendicontazione: 2019-07-01 al 2021-06-30
Introduction, objectives and importance
Stem cells play a fundamental role in the maintenance of adult tissue architecture and integrity by providing a pool of differentiated cells through asymmetric division (1). Therefore, they must shield from endogenous threats, such as transposable elements, as well as from exogenous insults, e.g. genotoxic stress or viral infections. Early antiviral responses in mammals rely on the expression of type I and type III interferons (IFN), which act on differentiated cells in an autocrine and paracrine manner to promote the transcription of interferon stimulated genes (ISGs) that encode antiviral effector proteins (2). However, protection conferred by the IFN pathway is severely compromised in embryonic and adult stem cells, which lack key components of the pathway (3). IFN incompetence may ensure that stem cells are protected from the cytostatic properties of IFN, such as antiproliferative and proapoptotic effects. Despite their IFN unresponsiveness, stem cells are largely resistant to viral infection, which can be attributed in part to the steady-state expression of ISGs and other factors restricting infection (4). Nonetheless, immune responses in stem cells remain poorly characterised.
In invertebrates and plants, antiviral immunity relies on RNA interference (RNAi), which is initiated by the cleavage of viral double-stranded RNA (dsRNA) by a Dicer protein, generating small interfering RNAs (siRNAs) which guide the degradation of viral RNA (3). Irrespective of infection, RNAi also has a role in regulating cellular gene expression via micro RNAs (miRNAs) produced by Dicer through cleavage of pre-miRNAs. Organisms that use RNAi both as an antiviral response and as a way of regulating mRNA translation with miRNAs have several Dicer genes, the product of which are dedicated to processing either dsRNA or pre-miRNAs (5). In mammals, a single Dicer gene has been described, which encodes a Dicer protein that generates miRNAs but cleaves dsRNA only poorly (6). Whether antiviral RNAi exists in mammalian cells and is relevant to immunity remains highly controversial (3).
This project aims at delineating the role and importance of antiviral RNAi in mammals, by answering three mains questions: where does antiviral RNAi happen in vivo, how does it happen, and what is its importance in antiviral immunity compare to IFN activation. Understanding the role and importance of antiviral RNAi in mammals will advance basic knowledge of mammalian immunity, and could also pave the way to innovative antiviral therapies in humans.
Conclusions of the action
An novel isoform of Dicer, termed antiviral Dicer (aviD) was discovered in the course of this work. This isoform is expressed in mouse and human stem cells within tissues, and protects them against viruses such as Zika virus or severe acute respiratory syndrome coronavirus 2, by implementing canonical antiviral RNAi.
References
1. E. Batlle, H. Clevers, Nat. Med. 23, 1124–1134 (2017).
2. X. Tan, L. Sun, J. Chen, Z. J. Chen, Annu. Rev. Microbiol. 72, 447–478 (2018).
3. P. V. Maillard, A. G. Veen, E. Z. Poirier, C. Reis e Sousa, EMBO J. 38 (2019), doi:10.15252/embj.2018100941.
4. X. Wu et al., Cell (2017), doi:10.1016/j.cell.2017.11.018.
5. S.-W. Ding, Nat. Rev. Immunol. 10, 632–644 (2010).
6. E. Ma, I. J. MacRae, J. F. Kirsch, J. A. Doudna, J. Mol. Biol. 380, 237–243 (2008).
Stem cells play a fundamental role in the maintenance of adult tissue architecture and integrity by providing a pool of differentiated cells through asymmetric division (1). Therefore, they must shield from endogenous threats, such as transposable elements, as well as from exogenous insults, e.g. genotoxic stress or viral infections. Early antiviral responses in mammals rely on the expression of type I and type III interferons (IFN), which act on differentiated cells in an autocrine and paracrine manner to promote the transcription of interferon stimulated genes (ISGs) that encode antiviral effector proteins (2). However, protection conferred by the IFN pathway is severely compromised in embryonic and adult stem cells, which lack key components of the pathway (3). IFN incompetence may ensure that stem cells are protected from the cytostatic properties of IFN, such as antiproliferative and proapoptotic effects. Despite their IFN unresponsiveness, stem cells are largely resistant to viral infection, which can be attributed in part to the steady-state expression of ISGs and other factors restricting infection (4). Nonetheless, immune responses in stem cells remain poorly characterised.
In invertebrates and plants, antiviral immunity relies on RNA interference (RNAi), which is initiated by the cleavage of viral double-stranded RNA (dsRNA) by a Dicer protein, generating small interfering RNAs (siRNAs) which guide the degradation of viral RNA (3). Irrespective of infection, RNAi also has a role in regulating cellular gene expression via micro RNAs (miRNAs) produced by Dicer through cleavage of pre-miRNAs. Organisms that use RNAi both as an antiviral response and as a way of regulating mRNA translation with miRNAs have several Dicer genes, the product of which are dedicated to processing either dsRNA or pre-miRNAs (5). In mammals, a single Dicer gene has been described, which encodes a Dicer protein that generates miRNAs but cleaves dsRNA only poorly (6). Whether antiviral RNAi exists in mammalian cells and is relevant to immunity remains highly controversial (3).
This project aims at delineating the role and importance of antiviral RNAi in mammals, by answering three mains questions: where does antiviral RNAi happen in vivo, how does it happen, and what is its importance in antiviral immunity compare to IFN activation. Understanding the role and importance of antiviral RNAi in mammals will advance basic knowledge of mammalian immunity, and could also pave the way to innovative antiviral therapies in humans.
Conclusions of the action
An novel isoform of Dicer, termed antiviral Dicer (aviD) was discovered in the course of this work. This isoform is expressed in mouse and human stem cells within tissues, and protects them against viruses such as Zika virus or severe acute respiratory syndrome coronavirus 2, by implementing canonical antiviral RNAi.
References
1. E. Batlle, H. Clevers, Nat. Med. 23, 1124–1134 (2017).
2. X. Tan, L. Sun, J. Chen, Z. J. Chen, Annu. Rev. Microbiol. 72, 447–478 (2018).
3. P. V. Maillard, A. G. Veen, E. Z. Poirier, C. Reis e Sousa, EMBO J. 38 (2019), doi:10.15252/embj.2018100941.
4. X. Wu et al., Cell (2017), doi:10.1016/j.cell.2017.11.018.
5. S.-W. Ding, Nat. Rev. Immunol. 10, 632–644 (2010).
6. E. Ma, I. J. MacRae, J. F. Kirsch, J. A. Doudna, J. Mol. Biol. 380, 237–243 (2008).
I identified a new Dicer isoform produced from the human and mouse Dicer gene that is better able to process dsRNA than canonical Dicer. This isoform, termed antiviral Dicer (aviD) is generated by alternative splicing of Dicer mRNA and lacks the central Hel2i domain of the helicase present in canonical Dicer (referred to as Dicer hereafte). aviD expression is enriched in stem cells within adult tissues, including gut, skin and brain. Using a model of brain organoid infected with Zika virus (ZIKV) or SARS-CoV-2, I demonstrated that aviD protects adult stem cells from RNA viruses by mounting a canonical antiviral RNAi response. This work highlights the composite nature of antiviral immunity in mammals, tailored to the differentiation status of the cell. This work was published as a scientific article in 2021 (Poirier et al., Science) and presented at various scientific workshops and symposium, including the SY-Stem Symposia (2021).
The discovery of the role and importance of aviD-driven antiviral immunity in mammals greatly advances our understanding of mammalian immunity, specifically regarding the antiviral resistance of stem cells. In the context of the coronavirus pandemics, it is interesting to note that aviD displays antiviral action against SARS-CoV-2, protecting stem cells against the virus. Whether certain patients display decrease aviD activity, leading to increased stem cell infection and exacerbated pathology, remains to be determined. The discovery of aviD overall opens new avenues of research in the field of antiviral immunity and paves the way to new applications in the clinic.