Nuclear Phosphoinositide Signalling Coupled Variant Poly(A) Polymerase Star‐PAP Regulates Metastatic Invasion
Authors: Ap Sudheesh , Laishram Rakesh Singh 
Published: November 2023

mRNA generation in eukaryotes involves multiple steps from transcription to the processing at the 3′‐untranslated region (UTR). 3′‐end processing involves two tightly coupled steps: endonucleolytic cleavage followed by addition of a poly(A) tail (polyadenylation) at the 3′‐UTR. Polyadenylation is essential for stability and translation efficiency of the mRNA and is carried out by enzymes called poly(A) polymerases (PAPs) in a large multiprotein complex that includes cleavage and polyadenylation factors such as CPSF, CstF, CFIm, IIm, Symplekin, and PABP. Star‐PAP is a non‐canonical PAP regulated by nuclear phosphoinositide second messenger PI4,5P2 (PIP2) and the enzyme Type I phosphatidylinositol 4‐phosphate 5‐kinase alpha (PIPKIα). It selects mRNA targets for polyadenylation such as those involved in critical cellular functions like oxidative stress response and apoptosis. Analysis of earlier Star‐PAP microarray indicated a large proportion of Star‐PAP regulated mRNAs involved in cancer metastasis suggesting a putative role of Star‐PAP mediated 3′‐end processing in metastasis. Some of the target mRNAs such as KISS1R, NME1, CDH1, CDH13, FEZ1, and WIF1 encode anti‐metastatic factors in breast cancer. We confirmed the regulation of these target mRNAs by both Star‐PAP and its co‐regulator PIPKIα knockdown individually or together, which resulted in compromised cleavage and polyadenylation of these mRNAs.

Examining various metastatic cells showed that Star‐PAP protein levels correspond to the invasive potential of cells. MDA‐MB‐231 (metastatic breast cancer cell line) with high invasive potential had negligible Star‐PAP level while MCF7 (a non‐metastatic breast cancer cell line) with low invasiveness or normal MCF10 cells showed high endogenous Star‐PAP level. Concomitantly, silencing of Star‐PAP increased the invasiveness in MCF7 cell line while overexpression of Star‐PAP in MDA‐MB‐231 decreased the invasiveness in wound healing assays indicating the role of Star‐PAP as a negative regulator of metastatic invasion. To further test this, constructs expressing different levels of Star‐PAP was created by introducing mutations in the promoter of Star‐PAP expressing plasmid. Rescue experiment with transient expression of different Star‐PAP expressing mutants in MDA‐MB‐231 resulted in a progressive decrease in the invasiveness corresponding to the relative Star‐PAP levels in the cell. Interestingly, knockdown of Star‐PAP co‐regulator, PIPKIα in MCF7 or its overexpression in MDA‐MB‐231 cells also had similar effects as Star‐PAP indicating the significance of Star‐PAP‐PIPKIα interaction in cell invasion. This was further confirmed using PIPKIα non‐interacting mutant of Star‐PAP (S6A) or Star‐PAP N‐terminal deletion. Unlike the wild type, both mutants did not show any significant alteration in the invasiveness of MDA‐MB‐231 compared to wild type. Further using in vivo approach, we have demonstrated that MDA‐MB‐231 mediated tumor formation in BALB/c SCID mice was rescued by overexpression of Star‐PAP. Our study shows Star‐PAP as an anti‐invasive factor and establishes Star‐PAP and PIPKIα mediated 3′‐end processing as a key regulatory mechanism in the metastatic invasion.