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Most circular RNAs in Life Science come from the reverse splicing of exons and form a closed loop structure with covalent bonds
.
Recently, more and more researches have focused on the characteristics and biological functions of circular RNA, including its involvement in regulating signal transduction, cell proliferation and innate immune response (see the review Nat Rev Mol Cell Biol 2020 for details)
.
It is worth mentioning that the high stability of circular RNAs, which are different from the folded conformation of linear RNAs, give them unique application potential.
For example, they can be used as translation vectors for the expression of functional proteins, or as nucleic acid aptamers to regulate natural immune reactions
.
On December 23, 2021, Cell Press, the journal Molecular Cell, published online the latest research progress of circular RNA by Chen Lingling's research group at the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences "RNA circles with" minimized immunogenicity as potent PKR inhibitors"
.
This work clarified the problem of the immunogenicity of synthesized circular RNA in vitro, and found that non-immunogenic circular RNA with a double-stranded structure can effectively inhibit the activation of natural immune factor PKR, providing future intervention and treatment of autoimmune diseases using circular RNA A whole new way
.
Long press the picture to identify the QR code to read the original text.
It should be pointed out that recent studies have found that the circular RNA synthesized in vitro can be recognized by the innate immune receptor RIG-I, and then cause innate immune response (Chen, Mol Cell 2017; Chen, Mol Cell 2019); on the other hand, some researchers believe that the circular RNA synthesized in vitro is not immunogenic, and the natural immune response observed before is caused by linear RNA contamination in the synthesized circular RNA (Wesselhoeft, Mol Cell 2019)
.
Therefore, elucidating the synthesis pathway of circular RNA and the immunogenicity of its products are crucial for its future applications
.
At present, there are two main ways to synthesize circular RNA in vitro: catalyzed by T4 RNA ligase to form a loop, and type I intron ribozyme self-splicing to form a loop
.
By synthesizing circular RNA in vitro and comparing its immunogenicity in the above manner, Chen Lingling's team found that circular RNA synthesized by ligase does not cause innate immune response in cells, but circular RNA formed by self-splicing of two different type I introns.
Response
.
After comparative analysis, the researchers found that only 1 to 3 exogenous nucleotides were introduced based on the ligase-forming loop, but the self-splicing loop of the intron would introduce 74 or 186 exogenous nucleotides.
These exogenous sequences The introduction of the target circular RNA changes the folding, indicating that the exogenous sequence introduced by the self-splicing of the type I intron into a loop is one of the reasons for the natural immune response
.
The previous work of Chen Lingling's research group found that endogenous circular RNA containing 16-26bp double-stranded structure can inhibit the abnormal activation of immune factor PKR (Liu, Cell 2019), suggesting that in vitro synthesis of non-immunogenic circular RNA containing short double-stranded structure is expected Become a new method to treat autoimmune diseases related to excessive activation of PKR
.
Researchers found that circular RNA synthesized by RNA ligase not only has low immunogenicity, but also retains a 16-26bp double-stranded structure, which can inhibit the activation of PKR, and has an inhibitory effect that is about a thousand times greater than that of PKR small molecule inhibitors
.
Dr.
Liu Chuxiao, postdoctoral fellow of Chen Lingling's research group at the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology), Chinese Academy of Sciences, and Guo Sikun, a doctoral candidate, are the co-first authors of the paper, and researcher Chen Lingling is the corresponding author of the paper
.
This work was supported by researcher Yang Li from the Shanghai Institute of Nutrition and Health of the Chinese Academy of Sciences, PhD student Xu Yifeng in the Chen Lingling group, and Dr.
Nan Fang from the Yang Li group.
Support
.
Interview with Cell Press Cell Press on the immunogenicity and potential application patterns of in vitro synthesized circular RNA.
The author specially invited researcher Chen Lingling to conduct an interview on behalf of the research team, and asked her to further explain it in detail
.
CellPress: How did you come up with synthetic RNA loops as PKR inhibitors? Researcher Chen Lingling: The previous work of our research group found that the endogenous circular RNA containing 16-33bp double-stranded structure can effectively inhibit the activation of the natural immune factor PKR, participate in the regulation of natural immune response, and is closely related to human diseases such as autoimmune diseases (Liu, Cell 2019)
.
This work suggests that the circular RNA synthesized in vitro is expected to be used to treat autoimmune diseases related to abnormal activation of PKR
.
Therefore, we explored different ways of synthesizing circular RNA in vitro and the immunogenicity of its products
.
Through in-depth analysis of the characteristics of different methods of in vitro synthesis of circular RNAs, we have obtained circular RNAs with extremely low immunogenicity and containing 16-26bp double-stranded structures, which can be used for future interventions in inflammatory autoimmune diseases using such new biomolecules The treatment puts forward new ideas
.
CellPress: How is the immunogenicity of circular RNA synthesized from self-splicing introns derived from T4 bacteriophage and Anabaena (Ana) tested for its immunogenicity? Researcher Chen Lingling: For the circular RNA produced by different synthetic methods, we mainly detect the immunogenicity by transfecting the circular RNA synthesized in vitro into the cell
.
This includes detection of the expression levels of immune factors IFNβ, IL6, TNFα and immune recognition receptor RIG-I at the mRNA level by fluorescent quantitative PCR (qPCR), and at the protein level, secreted by enzyme-linked immunosorbent assay (ELISA) Changes in cytokines such as IFNβ and IL6 in the extracellular matrix
.
In the future, we also need to do more and more detailed circular RNA immunogenicity tests at the level of model animals, such as testing on mouse models, including model animals' overall inflammatory immune response testing and testing of different immune cell subtypes
.
CellPress: Which areas of the RNA loop synthesized by T4 RNA ligase can inhibit the activity of PKR? Researcher Chen Lingling: We analyzed the secondary structure of circular RNA synthesized in vitro through experimental analysis of circular RNA synthesized in vitro, and found that circular RNA synthesized by T4 RNA ligase retains a short double-stranded structure similar to endogenous RNA
.
By analyzing the folded conformation of circular RNA synthesized in vitro after binding to PKR, compared with before PKR binding, we found that there is a significant reduction in the SHAPE label signal near the double-stranded structure region of circular RNA, which further shows that the short double-stranded structure of circular RNA is similar to PKR.
Interaction
.
In short, because the circular RNA synthesized in vitro (such as circular POLR2A, circular CAMSAP1, etc.
) contains a short double-stranded structure, it can inhibit the activation of PKR in vitro and in cells
.
CellPress: What is the PKR inhibitory effect of the circular RNA found in this study? How can this help the treatment of PKR-related autoimmune diseases? Researcher Chen Lingling: Through related experiments, we found that circular RNA synthesized by this type of T4 RNA ligase can effectively inhibit PKR activation in vitro and in cells, and is more than a thousand times higher than the commonly used PKR small molecule inhibitors 2-AP and C16 Better suppression effect
.
Related research lays an important foundation for the further application of circular RNA synthesized in vitro, and provides a new idea for the intervention and treatment of autoimmune diseases using circular RNA
.
At the same time, the study of how to obtain low-immunogenic circular RNA and its mechanism will also bring promising prospects for the establishment of a new platform for gene expression based on circular RNA technology
.
CellPress: What is the focus of your next work? Researcher Chen Lingling: Our next work focus is mainly divided into three areas: First, continue to develop new methods for studying the function and regulation of circular RNA, discover new functions of circular RNA and clarify the molecular mechanism of functional circular RNA in regulation; It is to improve the method of in vitro synthesis of circular RNA, increase the efficiency of in vitro synthesis of non-immunogenic circular RNA, and expand the application scenarios of in vitro synthesis of circular RNA; the third is to use disease animal models to explore the use of circular RNA for inflammatory autoimmune disease intervention and treatment Possibility
.
Brief introduction to the corresponding author Lingling Chen Researcher Lingling Chen is the research team leader of the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology), Chinese Academy of Sciences
.
He has been engaged in basic research on RNA metabolism and function for a long time, creating and using new technology systems for RNA research, discovering new families of non-coding RNAs and their ubiquitous presence in mammalian cells, analyzing their production pathways and revealing their important functions
.
Related work has been published in journals such as Cell and Science
.
Currently serving as an editorial board member of journals such as Cell, Science, Mol Cell, etc.
.
In 2017, he was selected as the HHMI International Research Scholar and the National Natural Science Foundation of China for Outstanding Youth Science Fund; he was awarded the China Youth Science and Technology Award (Special Award), Scientific Exploration Award, and Mid-Career Research Award from the International RNA Society
.
The research results of related paper information are published in the Molecular Cell journal under Cell Press.
Click "Read Full Text" or scan the QR code below to view the paper
.
▌Paper title: RNA circles with minimized immunogenicity as potent PKR inhibitors▌Paper URL: https://▌DOI: https://doi .
org/10.
1016/j.
molcel.
2021.
11.
019 Long press the picture to identify the QR code to read the original text.
In 1974, we published the first flagship journal "Cell"
.
Today, CellPress has developed into an international frontier academic publishing house with more than 50 journals in the field of science
.
We firmly believe that the power of science will always benefit mankind
.
Recommended reading by CellPress Cell Press.
Xiao Wuhan's team reveals a new molecular mechanism for precise regulation of innate immune response | Cell Press Dialogue Scientist ▲
.
Recently, more and more researches have focused on the characteristics and biological functions of circular RNA, including its involvement in regulating signal transduction, cell proliferation and innate immune response (see the review Nat Rev Mol Cell Biol 2020 for details)
.
It is worth mentioning that the high stability of circular RNAs, which are different from the folded conformation of linear RNAs, give them unique application potential.
For example, they can be used as translation vectors for the expression of functional proteins, or as nucleic acid aptamers to regulate natural immune reactions
.
On December 23, 2021, Cell Press, the journal Molecular Cell, published online the latest research progress of circular RNA by Chen Lingling's research group at the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences "RNA circles with" minimized immunogenicity as potent PKR inhibitors"
.
This work clarified the problem of the immunogenicity of synthesized circular RNA in vitro, and found that non-immunogenic circular RNA with a double-stranded structure can effectively inhibit the activation of natural immune factor PKR, providing future intervention and treatment of autoimmune diseases using circular RNA A whole new way
.
Long press the picture to identify the QR code to read the original text.
It should be pointed out that recent studies have found that the circular RNA synthesized in vitro can be recognized by the innate immune receptor RIG-I, and then cause innate immune response (Chen, Mol Cell 2017; Chen, Mol Cell 2019); on the other hand, some researchers believe that the circular RNA synthesized in vitro is not immunogenic, and the natural immune response observed before is caused by linear RNA contamination in the synthesized circular RNA (Wesselhoeft, Mol Cell 2019)
.
Therefore, elucidating the synthesis pathway of circular RNA and the immunogenicity of its products are crucial for its future applications
.
At present, there are two main ways to synthesize circular RNA in vitro: catalyzed by T4 RNA ligase to form a loop, and type I intron ribozyme self-splicing to form a loop
.
By synthesizing circular RNA in vitro and comparing its immunogenicity in the above manner, Chen Lingling's team found that circular RNA synthesized by ligase does not cause innate immune response in cells, but circular RNA formed by self-splicing of two different type I introns.
Response
.
After comparative analysis, the researchers found that only 1 to 3 exogenous nucleotides were introduced based on the ligase-forming loop, but the self-splicing loop of the intron would introduce 74 or 186 exogenous nucleotides.
These exogenous sequences The introduction of the target circular RNA changes the folding, indicating that the exogenous sequence introduced by the self-splicing of the type I intron into a loop is one of the reasons for the natural immune response
.
The previous work of Chen Lingling's research group found that endogenous circular RNA containing 16-26bp double-stranded structure can inhibit the abnormal activation of immune factor PKR (Liu, Cell 2019), suggesting that in vitro synthesis of non-immunogenic circular RNA containing short double-stranded structure is expected Become a new method to treat autoimmune diseases related to excessive activation of PKR
.
Researchers found that circular RNA synthesized by RNA ligase not only has low immunogenicity, but also retains a 16-26bp double-stranded structure, which can inhibit the activation of PKR, and has an inhibitory effect that is about a thousand times greater than that of PKR small molecule inhibitors
.
Dr.
Liu Chuxiao, postdoctoral fellow of Chen Lingling's research group at the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology), Chinese Academy of Sciences, and Guo Sikun, a doctoral candidate, are the co-first authors of the paper, and researcher Chen Lingling is the corresponding author of the paper
.
This work was supported by researcher Yang Li from the Shanghai Institute of Nutrition and Health of the Chinese Academy of Sciences, PhD student Xu Yifeng in the Chen Lingling group, and Dr.
Nan Fang from the Yang Li group.
Support
.
Interview with Cell Press Cell Press on the immunogenicity and potential application patterns of in vitro synthesized circular RNA.
The author specially invited researcher Chen Lingling to conduct an interview on behalf of the research team, and asked her to further explain it in detail
.
CellPress: How did you come up with synthetic RNA loops as PKR inhibitors? Researcher Chen Lingling: The previous work of our research group found that the endogenous circular RNA containing 16-33bp double-stranded structure can effectively inhibit the activation of the natural immune factor PKR, participate in the regulation of natural immune response, and is closely related to human diseases such as autoimmune diseases (Liu, Cell 2019)
.
This work suggests that the circular RNA synthesized in vitro is expected to be used to treat autoimmune diseases related to abnormal activation of PKR
.
Therefore, we explored different ways of synthesizing circular RNA in vitro and the immunogenicity of its products
.
Through in-depth analysis of the characteristics of different methods of in vitro synthesis of circular RNAs, we have obtained circular RNAs with extremely low immunogenicity and containing 16-26bp double-stranded structures, which can be used for future interventions in inflammatory autoimmune diseases using such new biomolecules The treatment puts forward new ideas
.
CellPress: How is the immunogenicity of circular RNA synthesized from self-splicing introns derived from T4 bacteriophage and Anabaena (Ana) tested for its immunogenicity? Researcher Chen Lingling: For the circular RNA produced by different synthetic methods, we mainly detect the immunogenicity by transfecting the circular RNA synthesized in vitro into the cell
.
This includes detection of the expression levels of immune factors IFNβ, IL6, TNFα and immune recognition receptor RIG-I at the mRNA level by fluorescent quantitative PCR (qPCR), and at the protein level, secreted by enzyme-linked immunosorbent assay (ELISA) Changes in cytokines such as IFNβ and IL6 in the extracellular matrix
.
In the future, we also need to do more and more detailed circular RNA immunogenicity tests at the level of model animals, such as testing on mouse models, including model animals' overall inflammatory immune response testing and testing of different immune cell subtypes
.
CellPress: Which areas of the RNA loop synthesized by T4 RNA ligase can inhibit the activity of PKR? Researcher Chen Lingling: We analyzed the secondary structure of circular RNA synthesized in vitro through experimental analysis of circular RNA synthesized in vitro, and found that circular RNA synthesized by T4 RNA ligase retains a short double-stranded structure similar to endogenous RNA
.
By analyzing the folded conformation of circular RNA synthesized in vitro after binding to PKR, compared with before PKR binding, we found that there is a significant reduction in the SHAPE label signal near the double-stranded structure region of circular RNA, which further shows that the short double-stranded structure of circular RNA is similar to PKR.
Interaction
.
In short, because the circular RNA synthesized in vitro (such as circular POLR2A, circular CAMSAP1, etc.
) contains a short double-stranded structure, it can inhibit the activation of PKR in vitro and in cells
.
CellPress: What is the PKR inhibitory effect of the circular RNA found in this study? How can this help the treatment of PKR-related autoimmune diseases? Researcher Chen Lingling: Through related experiments, we found that circular RNA synthesized by this type of T4 RNA ligase can effectively inhibit PKR activation in vitro and in cells, and is more than a thousand times higher than the commonly used PKR small molecule inhibitors 2-AP and C16 Better suppression effect
.
Related research lays an important foundation for the further application of circular RNA synthesized in vitro, and provides a new idea for the intervention and treatment of autoimmune diseases using circular RNA
.
At the same time, the study of how to obtain low-immunogenic circular RNA and its mechanism will also bring promising prospects for the establishment of a new platform for gene expression based on circular RNA technology
.
CellPress: What is the focus of your next work? Researcher Chen Lingling: Our next work focus is mainly divided into three areas: First, continue to develop new methods for studying the function and regulation of circular RNA, discover new functions of circular RNA and clarify the molecular mechanism of functional circular RNA in regulation; It is to improve the method of in vitro synthesis of circular RNA, increase the efficiency of in vitro synthesis of non-immunogenic circular RNA, and expand the application scenarios of in vitro synthesis of circular RNA; the third is to use disease animal models to explore the use of circular RNA for inflammatory autoimmune disease intervention and treatment Possibility
.
Brief introduction to the corresponding author Lingling Chen Researcher Lingling Chen is the research team leader of the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology), Chinese Academy of Sciences
.
He has been engaged in basic research on RNA metabolism and function for a long time, creating and using new technology systems for RNA research, discovering new families of non-coding RNAs and their ubiquitous presence in mammalian cells, analyzing their production pathways and revealing their important functions
.
Related work has been published in journals such as Cell and Science
.
Currently serving as an editorial board member of journals such as Cell, Science, Mol Cell, etc.
.
In 2017, he was selected as the HHMI International Research Scholar and the National Natural Science Foundation of China for Outstanding Youth Science Fund; he was awarded the China Youth Science and Technology Award (Special Award), Scientific Exploration Award, and Mid-Career Research Award from the International RNA Society
.
The research results of related paper information are published in the Molecular Cell journal under Cell Press.
Click "Read Full Text" or scan the QR code below to view the paper
.
▌Paper title: RNA circles with minimized immunogenicity as potent PKR inhibitors▌Paper URL: https://▌DOI: https://doi .
org/10.
1016/j.
molcel.
2021.
11.
019 Long press the picture to identify the QR code to read the original text.
In 1974, we published the first flagship journal "Cell"
.
Today, CellPress has developed into an international frontier academic publishing house with more than 50 journals in the field of science
.
We firmly believe that the power of science will always benefit mankind
.
Recommended reading by CellPress Cell Press.
Xiao Wuhan's team reveals a new molecular mechanism for precise regulation of innate immune response | Cell Press Dialogue Scientist ▲
