A Potential Effect of Cordycepin on the Feedback between Polyadenylation and PI3K/Akt/mTOR Signalling

Polyadenylation is a key step in mRNA maturation and leads to the sequential addition of adenosines forming a poly(A) tail, which is important in mRNA stability, nuclear export, transcription termination, and translational control. This process requires RNA-binding proteins, such as polyadenylation machinery, in pre-mRNA 3’processing multiprotein complexes. Cordycepin (3’-deoxyadenosine) is a natural compound known to metabolise to cordycepin triphosphate (CoTP), which once incorporated into the poly(A) tail, causes chain termination and is thought to restrict the dissociation of polyadenylation machinery such as WDR33. There are known effects of cordycepin on inflammation, cancer progression, and signal transduction pathways, such as PI3K/Akt/mTOR signalling, however the clear mechanism of action of cordycepin is still elusive. This study analysed multiple microarray and RNA-Seq datasets with cordycepin treatment and found that cordycepin had a similar effect on gene expression to the known PI3K inhibitor, LY294002. Consistently, differentially expressed genes with cordycepin treatment were also linked with repression of PI3K/Akt signalling. Furthermore, through western blotting, cordycepin and PI3K inhibition was found to repress phosphorylation of kinases downstream to AKT and mTORC1, in RAW264.7 macrophages and MCF-7 cells, and phosphorylated AMPK (Thr172) in RAW264.7 macrophages only. These effects demonstrate that cordycepin can repress AKT and mTORC1 probably by inhibiting PI3K. Through Ingenuity Pathway Analysis, cordycepin treatment was found to have opposite effects to gene expression to the stimulation and activation of upstream regulators such as LPS, TNF, multiple interleukins, EGF, TGF-β, PDGF, and their respective receptors. In RAW264.7 macrophages, differentially expressed genes with cordycepin treatment showed repression of NF-κB signalling, and immunofluorescence confirmed that cordycepin represses LPS-induced NF-κB (p65) nuclear translocation. Also, cordycepin treatment repressed relative mRNA expression of both inflammatory mRNA markers in RAW264.7 macrophages, and growth factor-dependent mRNA markers and transcriptions factors, such as MYC and JUN in MCF-7 cells. Cordycepin treatment prior to EGF stimulation in HEK293 cells also repressed relative mRNA expression of immediate early genes (IEG’s), c-MYC and c-JUN through qPCR. This altogether showed that cordycepin represses gene expression downstream from inflammatory and growth factor responses via inhibiting activity of signalling pathways and transcription factors. AMPK has previously been suggested to be the key mechanistic target of cordycepin. However, this study has shown that cordycepin can still repress mRNA expression of IEG’s and biological pathways such as chromatin remodelling and transcription in CRISPR-Cas9 AMPK knockout HEK293 cells with EGF stimulation. Cordycepin also repressed genes linked to multiple inositol phosphate metabolic pathways in the presence of growth factors in MCF-7, MDA-MB-231 cells, NIH3T3 fibroblasts, and in CRISPR-Cas9 AMPK Knockout HEK293 cells. This included key kinases important for generating inositol phosphates upstream to PI3K signalling, indicating that cordycepin can still affect PI3K activity in the absence of AMPK. Label-free quantitative proteomics of Orthogonal Organic Phase Separation (OOPS) fractions showed that both cordycepin and LY294002 treatments can shift RBP’s, including polyadenylation factors, towards the RNA-bound Interphase. PI3K inhibition had a more substantial effect than cordycepin treatment and shifted WDR33 towards the Interphase suggesting that PI3K inhibition traps WDR33 on RNA, similarly to CoTP. Knockdown of WDR33 in RAW264.7 macrophages was found to repress PI3K/Akt/mTOR signalling through phosphorylating AMPK (Thr172), and repressing GSK3β (Ser9), 4E-BP1 (Thr37/46) phosphorylation. Coupled together, this study suggests that there is a feedback effect between PI3K and WDR33. The findings in this study therefore both complement known effects of cordycepin on inflammatory, and growth factor stimulation, shows that cordycepin probably acts through PI3K inhibition, and suggesting that cordycepin may act through affecting the feedback between PI3K and the polyadenylation machinery

A Potential Effect of Cordycepin on the Feedback between Polyadenylation and PI3K/Akt/mTOR Signalling

Polyadenylation is a key step in mRNA maturation and leads to the sequential addition of adenosines forming a poly(A) tail, which is important in mRNA stability, nuclear export, transcription termination, and translational control. This process requires RNA-binding proteins, such as polyadenylation machinery, in pre-mRNA 3’processing multiprotein complexes. Cordycepin (3’-deoxyadenosine) is a natural compound known to metabolise to cordycepin triphosphate (CoTP), which once incorporated into the poly(A) tail, causes chain termination and is thought to restrict the dissociation of polyadenylation machinery such as WDR33. There are known effects of cordycepin on inflammation, cancer progression, and signal transduction pathways, such as PI3K/Akt/mTOR signalling, however the clear mechanism of action of cordycepin is still elusive. This study analysed multiple microarray and RNA-Seq datasets with cordycepin treatment and found that cordycepin had a similar effect on gene expression to the known PI3K inhibitor, LY294002. Consistently, differentially expressed genes with cordycepin treatment were also linked with repression of PI3K/Akt signalling. Furthermore, through western blotting, cordycepin and PI3K inhibition was found to repress phosphorylation of kinases downstream to AKT and mTORC1, in RAW264.7 macrophages and MCF-7 cells, and phosphorylated AMPK (Thr172) in RAW264.7 macrophages only. These effects demonstrate that cordycepin can repress AKT and mTORC1 probably by inhibiting PI3K. Through Ingenuity Pathway Analysis, cordycepin treatment was found to have opposite effects to gene expression to the stimulation and activation of upstream regulators such as LPS, TNF, multiple interleukins, EGF, TGF-β, PDGF, and their respective receptors. In RAW264.7 macrophages, differentially expressed genes with cordycepin treatment showed repression of NF-κB signalling, and immunofluorescence confirmed that cordycepin represses LPS-induced NF-κB (p65) nuclear translocation. Also, cordycepin treatment repressed relative mRNA expression of both inflammatory mRNA markers in RAW264.7 macrophages, and growth factor-dependent mRNA markers and transcriptions factors, such as MYC and JUN in MCF-7 cells. Cordycepin treatment prior to EGF stimulation in HEK293 cells also repressed relative mRNA expression of immediate early genes (IEG’s), c-MYC and c-JUN through qPCR. This altogether showed that cordycepin represses gene expression downstream from inflammatory and growth factor responses via inhibiting activity of signalling pathways and transcription factors. AMPK has previously been suggested to be the key mechanistic target of cordycepin. However, this study has shown that cordycepin can still repress mRNA expression of IEG’s and biological pathways such as chromatin remodelling and transcription in CRISPR-Cas9 AMPK knockout HEK293 cells with EGF stimulation. Cordycepin also repressed genes linked to multiple inositol phosphate metabolic pathways in the presence of growth factors in MCF-7, MDA-MB-231 cells, NIH3T3 fibroblasts, and in CRISPR-Cas9 AMPK Knockout HEK293 cells. This included key kinases important for generating inositol phosphates upstream to PI3K signalling, indicating that cordycepin can still affect PI3K activity in the absence of AMPK. Label-free quantitative proteomics of Orthogonal Organic Phase Separation (OOPS) fractions showed that both cordycepin and LY294002 treatments can shift RBP’s, including polyadenylation factors, towards the RNA-bound Interphase. PI3K inhibition had a more substantial effect than cordycepin treatment and shifted WDR33 towards the Interphase suggesting that PI3K inhibition traps WDR33 on RNA, similarly to CoTP. Knockdown of WDR33 in RAW264.7 macrophages was found to repress PI3K/Akt/mTOR signalling through phosphorylating AMPK (Thr172), and repressing GSK3β (Ser9), 4E-BP1 (Thr37/46) phosphorylation. Coupled together, this study suggests that there is a feedback effect between PI3K and WDR33. The findings in this study therefore both complement known effects of cordycepin on inflammatory, and growth factor stimulation, shows that cordycepin probably acts through PI3K inhibition, and suggesting that cordycepin may act through affecting the feedback between PI3K and the polyadenylation machinery