{"title":"Advances in the elucidation of circRNA translocation","authors":"Yang Gu, Xiaoxue Zhou, Long Zhang","doi":"10.1002/mef2.87","DOIUrl":null,"url":null,"abstract":"<p>Recently a research article titled “Nuclear export of circular RNA” was published online in <i>Nature</i><span><sup>1</sup></span> as a collaboration between Ngo et al. This study revealed a distinct circular RNA (circRNA) transport mechanism compared to that of linear RNA and identified unique molecular pathways involved in circRNA transport, including key proteins such as Ran-GTP, IGF2BP1, and exportin-2.</p><p>CircRNA is a closed-loop structured noncoding RNA formed via pre-messenger RNA (mRNA) back-splicing. It regulates gene expression and participates in translation. CircRNA is associated with varieties of diseases, including those of the autoimmune, heart, liver, Alzheimer's, and cancer. It is important in cellular biology and disease research, and primarily exist in the cytoplasm; however, its translocation mechanism from the nucleus to cytoplasm remains unknown. In view of this, the research team conducted scientific research experiments.</p><p>In this study, the research team confirmed that most circRNAs were primarily located in the cytoplasm. They then depleted candidate proteins known to be involved in the transport of various linear RNA subtypes to examine whether the depletion would lead to circRNAs accumulation in the nucleus. The results showed that depletion of ALY, GANP, NXF1, UAP56, URH49, and exportin-5 did not affect circRNA transport. This indicated that bulk transport of circRNAs did not require a conventional mRNA export pathway. In contrast, they found that depleting the receptor CRM1 for ribosomal RNA and small nuclear RNA transport decreased nuclear circRNA content.<span><sup>2</sup></span> This unusual phenomenon attracted the attention of the research team. To further confirm this phenomenon, the cells were treated with the CRM1 inhibitor Selinexor, which also decreased the nuclear circRNA content and increased the cytoplasmic content.</p><p>The research team speculated that the CRM1 depletion could have been due to Ran-GTP consumption during nuclear transport complex assembly.<span><sup>3</sup></span> Therefore, CRM1 depletion could inhibit the assembly of the nuclear transport complex, thereby enhancing circRNA transport. Thus, they designed two experimental methods: measuring the nuclear and total cellular Ran content after CRM1 depletion using immunofluorescence and treating cells with sorbitol, a Ran-GTP inhibitor. These results confirmed that circRNA export depended on Ran-GTP.</p><p>The research team hypothesized that circRNA was transported via a transport protein under Ran-GTP-dependent conditions. Biotinylated SMARCA5 circRNA and linear RNA were used. The Ran content was controlled to identify protein exportin-2 using SMARCA5. Exportin-2 was then depleted in the cells, which increased nuclear circRNA content, but did not affect linear RNA. To ensure that the effect on circRNA export was not the result of the small interfering RNA (siRNA's) off-target effects, the depletion experiment was repeated with a second independent siRNA and CRISPR-Cas9 for targeted inactivation of the exportin-2 gene. To avoid the effects of circRNA with long half-lives, they labeled newly synthesized circRNAs with 4sU and found increased nuclear circRNA content and decreased cytoplasmic content after exportin-2 depletion.<span><sup>4</sup></span> Nuclear and cytoplasmic RNA samples from cells with and without exportin-2 depletion were subjected to RNA-seq analysis and single-molecule RNA in situ hybridization. These results indicated that circRNA was transported out of the nucleus by exportin-2. The research team's experimental data reveals that approximately 80% of the most abundantly expressed and likely functional circRNAs are transported by exportin-2.</p><p>During the experiments, the research team found that exportin-2 did not directly bind to circRNA, leading to the assumption that an adaptor aided in binding. To identify the proteins mediating exportin-2 and circRNA, a list of proteins identified by mass spectrometry were used and the proteins were captured using SMARCA5 circRNA. Eventually, 10 nuclear proteins that strongly bonded to circRNA in the presence of Ran-GTP were screened, including IGF2BP1 and IGF2BP2.<span><sup>5</sup></span> IGF2BP1 and IGF2BP2 were predicted affect circRNA transport. They attempted to prove this hypothesis via coimmunoprecipitation and direct depletion of these two proteins. IGF2BP1 and IGF2BP2 were found to directly bind to endogenous circRNA and mRNA. Depletion of IGF2BP1 and IGF2BP2 decreased circRNA expression levels, but did not strongly demonstrate their specific effects.</p><p>Next, the research team examined the collaboration between IGF2BP1 and exportin-2 for circRNA transport via testing their interaction in the cell nucleus. Coimmunoprecipitation and in vitro pull-down assays were used to reveal that exportin-2 directly interacted with IGF2BP1 through its RNA-binding domain. They then controlled Ran-GTP levels using the CRM1 inhibitor Selinexor in vivo and purified Ran-GTP in vitro to observe the interaction between IGF2BP1 and exportin-2. Additionally, a transport complex was constructed in vitro using recombinant full-length exportin-2, IGF2BP1, and Ran proteins, observing complex assembly under conditions with or without IGF2BP1 and different types of Rans (WT, Q69L, T24N). These studies showed that the formation of a transport complex by exportin-2, IGF2BP1, Ran-GTP, and circRNA was dependent on the binding of Ran and GTP. Subsequently, they confirmed circRNA binding to nuclear Ran using co-immunoprecipitation. A Ran-containing complex was confirmed to bind to circRNA in vivo from the Ran high-throughput sequencing results of RNA isolated by CLIP analysis. After analyzing the CLIP data, they discovered that IGF2BP1 depletion could hinder the binding between Ran and circRNA. Using co-immunoprecipitation and binding assay in vitro, they proved that IGF2BP1 was involved in the recruitment of Ran to RNA. The findings suggest that IGF2BP1 functions as an adapter during transport, mediating the assembly of a transport complex in the cell between exportin-2, Ran-GTP, and circRNA.</p><p>In summary, the research team first identified the Ran-GTP dependency of circRNA transport, then identified the Ran-GTP-dependent transport protein exportin-2, and finally the adaptor protein IGF2BP1. They confirmed that these proteins formed a transport complex through interaction and binding circRNA for transport, thus elucidating the transport pathway of circRNA (Figure 1). Although compelling evidence is currently lacking to prove that circRNA transport relies solely on Ran-GTP, exportin-2, and IGF2BP1, the study has demonstrated that circRNA transport is independent of the linear mRNA transport pathway. Notably, about 80% of the most abundantly expressed and functional circRNAs are transported by exportin-2, highlighting its significance for future research.</p><p>This article is crucial for advancing circRNA research and applications. First, the paper provides strong evidence that the nuclear export pathway of circRNA is independent of linear RNA, emphasizing the specialized transport mechanism involving specific proteins and Ran-GTP. This allows for the focused study of circRNA without affecting linear RNA functions and paves the way for developing small molecule drugs targeting circRNA. Second, circRNA is involved in various regulatory functions, such as acting as microRNA sponges, interacting with RNA-binding proteins, and modulating transcriptional activity. By understanding the export mechanism, researchers can better grasp how circRNA is positioned within the cell to exert these functions. Third, nuclear export is a pivotal step influencing circRNA stability and cytoplasmic localization, which are critical for their function. Abnormal circRNA expression and mislocalization are linked to various diseases. Insights from this study could lead to novel therapeutic strategies that target the nuclear export machinery to correct circRNA dysregulation.</p><p>Yang Gu wrote the manuscript and prepared the figure. Xiaoxue Zhou provided valuable discussion. Long Zhang approved the final version of the manuscript. All authors have read and approved the final manuscript.</p><p>The authors declare no conflict of interest.</p><p>Not applicable.</p>","PeriodicalId":74135,"journal":{"name":"MedComm - Future medicine","volume":"3 2","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/mef2.87","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"MedComm - Future medicine","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/mef2.87","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Recently a research article titled “Nuclear export of circular RNA” was published online in Nature1 as a collaboration between Ngo et al. This study revealed a distinct circular RNA (circRNA) transport mechanism compared to that of linear RNA and identified unique molecular pathways involved in circRNA transport, including key proteins such as Ran-GTP, IGF2BP1, and exportin-2.
CircRNA is a closed-loop structured noncoding RNA formed via pre-messenger RNA (mRNA) back-splicing. It regulates gene expression and participates in translation. CircRNA is associated with varieties of diseases, including those of the autoimmune, heart, liver, Alzheimer's, and cancer. It is important in cellular biology and disease research, and primarily exist in the cytoplasm; however, its translocation mechanism from the nucleus to cytoplasm remains unknown. In view of this, the research team conducted scientific research experiments.
In this study, the research team confirmed that most circRNAs were primarily located in the cytoplasm. They then depleted candidate proteins known to be involved in the transport of various linear RNA subtypes to examine whether the depletion would lead to circRNAs accumulation in the nucleus. The results showed that depletion of ALY, GANP, NXF1, UAP56, URH49, and exportin-5 did not affect circRNA transport. This indicated that bulk transport of circRNAs did not require a conventional mRNA export pathway. In contrast, they found that depleting the receptor CRM1 for ribosomal RNA and small nuclear RNA transport decreased nuclear circRNA content.2 This unusual phenomenon attracted the attention of the research team. To further confirm this phenomenon, the cells were treated with the CRM1 inhibitor Selinexor, which also decreased the nuclear circRNA content and increased the cytoplasmic content.
The research team speculated that the CRM1 depletion could have been due to Ran-GTP consumption during nuclear transport complex assembly.3 Therefore, CRM1 depletion could inhibit the assembly of the nuclear transport complex, thereby enhancing circRNA transport. Thus, they designed two experimental methods: measuring the nuclear and total cellular Ran content after CRM1 depletion using immunofluorescence and treating cells with sorbitol, a Ran-GTP inhibitor. These results confirmed that circRNA export depended on Ran-GTP.
The research team hypothesized that circRNA was transported via a transport protein under Ran-GTP-dependent conditions. Biotinylated SMARCA5 circRNA and linear RNA were used. The Ran content was controlled to identify protein exportin-2 using SMARCA5. Exportin-2 was then depleted in the cells, which increased nuclear circRNA content, but did not affect linear RNA. To ensure that the effect on circRNA export was not the result of the small interfering RNA (siRNA's) off-target effects, the depletion experiment was repeated with a second independent siRNA and CRISPR-Cas9 for targeted inactivation of the exportin-2 gene. To avoid the effects of circRNA with long half-lives, they labeled newly synthesized circRNAs with 4sU and found increased nuclear circRNA content and decreased cytoplasmic content after exportin-2 depletion.4 Nuclear and cytoplasmic RNA samples from cells with and without exportin-2 depletion were subjected to RNA-seq analysis and single-molecule RNA in situ hybridization. These results indicated that circRNA was transported out of the nucleus by exportin-2. The research team's experimental data reveals that approximately 80% of the most abundantly expressed and likely functional circRNAs are transported by exportin-2.
During the experiments, the research team found that exportin-2 did not directly bind to circRNA, leading to the assumption that an adaptor aided in binding. To identify the proteins mediating exportin-2 and circRNA, a list of proteins identified by mass spectrometry were used and the proteins were captured using SMARCA5 circRNA. Eventually, 10 nuclear proteins that strongly bonded to circRNA in the presence of Ran-GTP were screened, including IGF2BP1 and IGF2BP2.5 IGF2BP1 and IGF2BP2 were predicted affect circRNA transport. They attempted to prove this hypothesis via coimmunoprecipitation and direct depletion of these two proteins. IGF2BP1 and IGF2BP2 were found to directly bind to endogenous circRNA and mRNA. Depletion of IGF2BP1 and IGF2BP2 decreased circRNA expression levels, but did not strongly demonstrate their specific effects.
Next, the research team examined the collaboration between IGF2BP1 and exportin-2 for circRNA transport via testing their interaction in the cell nucleus. Coimmunoprecipitation and in vitro pull-down assays were used to reveal that exportin-2 directly interacted with IGF2BP1 through its RNA-binding domain. They then controlled Ran-GTP levels using the CRM1 inhibitor Selinexor in vivo and purified Ran-GTP in vitro to observe the interaction between IGF2BP1 and exportin-2. Additionally, a transport complex was constructed in vitro using recombinant full-length exportin-2, IGF2BP1, and Ran proteins, observing complex assembly under conditions with or without IGF2BP1 and different types of Rans (WT, Q69L, T24N). These studies showed that the formation of a transport complex by exportin-2, IGF2BP1, Ran-GTP, and circRNA was dependent on the binding of Ran and GTP. Subsequently, they confirmed circRNA binding to nuclear Ran using co-immunoprecipitation. A Ran-containing complex was confirmed to bind to circRNA in vivo from the Ran high-throughput sequencing results of RNA isolated by CLIP analysis. After analyzing the CLIP data, they discovered that IGF2BP1 depletion could hinder the binding between Ran and circRNA. Using co-immunoprecipitation and binding assay in vitro, they proved that IGF2BP1 was involved in the recruitment of Ran to RNA. The findings suggest that IGF2BP1 functions as an adapter during transport, mediating the assembly of a transport complex in the cell between exportin-2, Ran-GTP, and circRNA.
In summary, the research team first identified the Ran-GTP dependency of circRNA transport, then identified the Ran-GTP-dependent transport protein exportin-2, and finally the adaptor protein IGF2BP1. They confirmed that these proteins formed a transport complex through interaction and binding circRNA for transport, thus elucidating the transport pathway of circRNA (Figure 1). Although compelling evidence is currently lacking to prove that circRNA transport relies solely on Ran-GTP, exportin-2, and IGF2BP1, the study has demonstrated that circRNA transport is independent of the linear mRNA transport pathway. Notably, about 80% of the most abundantly expressed and functional circRNAs are transported by exportin-2, highlighting its significance for future research.
This article is crucial for advancing circRNA research and applications. First, the paper provides strong evidence that the nuclear export pathway of circRNA is independent of linear RNA, emphasizing the specialized transport mechanism involving specific proteins and Ran-GTP. This allows for the focused study of circRNA without affecting linear RNA functions and paves the way for developing small molecule drugs targeting circRNA. Second, circRNA is involved in various regulatory functions, such as acting as microRNA sponges, interacting with RNA-binding proteins, and modulating transcriptional activity. By understanding the export mechanism, researchers can better grasp how circRNA is positioned within the cell to exert these functions. Third, nuclear export is a pivotal step influencing circRNA stability and cytoplasmic localization, which are critical for their function. Abnormal circRNA expression and mislocalization are linked to various diseases. Insights from this study could lead to novel therapeutic strategies that target the nuclear export machinery to correct circRNA dysregulation.
Yang Gu wrote the manuscript and prepared the figure. Xiaoxue Zhou provided valuable discussion. Long Zhang approved the final version of the manuscript. All authors have read and approved the final manuscript.