Advances in the elucidation of circRNA translocation

Yang Gu, Xiaoxue Zhou, Long Zhang
{"title":"Advances in the elucidation of circRNA translocation","authors":"Yang Gu,&nbsp;Xiaoxue Zhou,&nbsp;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.

The authors declare no conflict of interest.

Not applicable.

Abstract Image

阐明 circRNA 转位的进展
此外,利用重组的全长 exportin-2、IGF2BP1 和 Ran 蛋白在体外构建了一个运输复合物,观察了在有或没有 IGF2BP1 和不同类型 Rans(WT、Q69L、T24N)的条件下复合物的组装情况。这些研究表明,exportin-2、IGF2BP1、Ran-GTP 和 circRNA 形成的运输复合物依赖于 Ran 和 GTP 的结合。随后,他们利用共沉淀免疫法证实了 circRNA 与核 Ran 的结合。通过CLIP分析分离出的RNA的Ran高通量测序结果证实,体内含有Ran的复合物与circRNA结合。在分析了CLIP数据后,他们发现IGF2BP1的缺失会阻碍Ran与circRNA的结合。通过共沉淀和体外结合试验,他们证明了IGF2BP1参与了Ran与RNA的招募。研究结果表明,IGF2BP1在转运过程中起着适配器的作用,介导细胞内exportin-2、Ran-GTP和circRNA之间转运复合物的组装。总之,研究小组首先确定了circRNA转运对Ran-GTP的依赖性,然后确定了依赖Ran-GTP的转运蛋白exportin-2,最后确定了适配器蛋白IGF2BP1。他们证实,这些蛋白通过相互作用形成转运复合物,并结合 circRNA 进行转运,从而阐明了 circRNA 的转运途径(图 1)。尽管目前还缺乏令人信服的证据证明 circRNA 的转运完全依赖于 Ran-GTP、exportin-2 和 IGF2BP1,但该研究证明 circRNA 的转运独立于 mRNA 的线性转运途径。值得注意的是,在表达量最高、功能最强的 circRNA 中,约有 80% 是通过 exportin-2 转运的,这凸显了 exportin-2 对未来研究的重要意义。首先,本文提供了强有力的证据,证明 circRNA 的核输出途径独立于线性 RNA,强调了涉及特定蛋白质和 Ran-GTP 的专门运输机制。这样就可以在不影响线性 RNA 功能的情况下对 circRNA 进行重点研究,并为开发以 circRNA 为靶标的小分子药物铺平了道路。其次,circRNA 参与多种调控功能,如充当 microRNA 海绵、与 RNA 结合蛋白相互作用以及调节转录活性。通过了解输出机制,研究人员可以更好地掌握 circRNA 如何在细胞内定位以发挥这些功能。第三,核输出是影响 circRNA 稳定性和细胞质定位的关键步骤,而细胞质定位对其功能至关重要。异常的 circRNA 表达和定位错误与多种疾病有关。这项研究的启示可能会带来针对核输出机制的新型治疗策略,以纠正circRNA的失调。Xiaoxue Zhou提供了宝贵的讨论意见。张龙批准了手稿的最终版本。所有作者均已阅读并批准最终稿件。作者声明无利益冲突。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
CiteScore
1.00
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信