What is Mechanism of Circular RNA Nuclear Export?

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What is Mechanism of Circular RNA Nuclear Export?

What is Mechanism of Circular RNA Nuclear Export?

Circular RNA (circRNA) was discovered over 20 years ago to be formed by precursor mRNA (pre-mRNA) through back splicing, with the ability to regulate gene expression and even participate in translation.

CircRNA has been associated with various diseases, including heart disease, liver disease, autoimmune diseases, Alzheimer’s disease, and cancer. While circRNA is mainly present in the cytoplasm, the process by which it is transported from the nucleus to the cytoplasm remains a mystery.

On February 14, 2024, a study titled “Nuclear export of circular RNA” was published online in the journal Nature by Vihandha Wickramasinghe’s team at the University of Melbourne, in collaboration with Gregory Goodall’s team at the University of Adelaide. The study revealed a different transport mechanism for circRNA compared to linear RNA, and identified a unique molecular pathway involved in circRNA transport, which includes key proteins such as Ran-GTP, IGF2BP1, and exportin-2.

What is Mechanism of Circular RNA Nuclear Export?

The research team first sequenced circRNA in the nucleus and cytoplasm, confirming that circRNA is mainly present in the cytoplasm. They then attempted to knock down several proteins involved in linear RNA transport (ALY, GANP, NXF1, UAP56, and URH49) and found that knocking down these proteins had no significant effect on circRNA transport. In contrast, knocking down CRM1, which is responsible for the forward transport of proteins, rRNA, and snRNA from the nucleus, significantly promoted the nuclear-cytoplasmic transport of circRNA, while having no effect on mRNA transport. Treatment of cells with the CRM1 inhibitor selinexor yielded similar results, further confirming the role of CRM1 in promoting circRNA transport.

Since the assembly of the nuclear transport complex requires Ran-GTP, the researchers speculated that inhibiting CRM1 might inhibit the formation of the transport complex, leading to the accumulation of more Ran-GTP in the nucleus, thereby aiding in circRNA transport. To validate this hypothesis, the researchers first confirmed by immunofluorescence that knocking down CRM1 significantly increased the levels of Ran protein in the nucleus. They then treated cells with the Ran-GTP inhibitor sorbitol and found that reducing Ran-GTP levels inhibited circRNA transport, while having no significant effect on mRNA distribution.

Considering that Ran-GTP mediates the transport of substances from the nucleus to the cytoplasm but does not directly transport RNA, the researchers hypothesized that there might be a transport protein similar to CRM1 that promotes circRNA transport in a manner dependent on Ran-GTP. To find such a protein, they used biotin-labeled, in vitro-synthesized circRNA (SMARCA5) to enrich for nuclear proteins that bind to it. Mass spectrometry analysis revealed that the nuclear transport protein exportin-2 was enriched by SMARCA5 and that exportin-2 also binds to Ran-GTP. Knocking down exportin-2 resulted in an increase in circRNA levels in the nucleus, with no significant decrease in the cytoplasm, possibly due to the high stability of circRNA, which is not easily degraded in a short period of time. Therefore, while knocking down exportin-2, the researchers also used 4sU labeling to mark newly synthesized circRNA and found, through qPCR, an increase in the amount of circRNA enriched in the nucleus and a corresponding decrease in the cytoplasm. Sequencing and fluorescence in situ hybridization (FISH) results also supported this finding, confirming that exportin-2 is a necessary protein for circRNA transport.

Although exportin-2 can transport circRNA, the researchers did not find that it directly binds to circRNA or mRNA, suggesting that there may be an adaptor protein that connects the two. Through mass spectrometry analysis, the researchers narrowed down the potential adaptors to 10 RNA-binding proteins, ultimately predicting that the two proteins involved in mRNA localization, IGF2BP1 and IGF2BP2, might affect circRNA transport. Evidence includes: 1) IGF2BP1 and IGF2BP2 can not only bind to SMARCA5, but Ran-GTP can also enhance their affinity; 2) Cross-linking immunoprecipitation (CLIP) results show that IGF2BP1 and IGF2BP2 can directly bind to endogenous circRNA and mRNA; 3) Direct knockdown of these two proteins affected the overall expression level of circRNA but did not directly determine their impact on transport.

Finally, to verify that exportin-2 and IGF2BP1 can jointly transport circRNA depending on Ran-GTP, the researchers first used in vitro-synthesized SMARCA5 to confirm through enrichment and in vitro reconstitution that exportin-2, IGF2BP1, Ran-GTP, and SMARCA5 can form a transport complex in vitro, and that the assembly of this complex depends on the binding of Ran and GTP. Subsequently, by using hits-CLIP to cross-link immunoprecipitation of nuclear Ran in vivo, it was confirmed that circRNA can directly bind to the complex containing Ran, and that the absence of IGF2BP1 weakens their binding. These results indicate that IGF2BP1 acts as an adaptor, mediating the formation of a transport complex between circRNA and Ran-GTP and exportin-2 in the cell, thereby promoting circRNA transport.

In summary, the researchers first discovered the influence of Ran-GTP on circRNA transport, identified the transport body exportin-2 and the adaptor protein IGF2BP1 dependent on Ran-GTP, verified that these proteins form a transport complex through interaction and bind circRNA for transport, and demonstrated a unique transport pathway different from linear mRNA.