Star-PAP RNA Binding Landscape Reveals Novel Role of Star-PAP in mRNA Metabolism That Requires RBM10-RNA Association

Abstract

Star-PAP is a non-canonical poly(A) polymerase that selects mRNA targets for polyadenylation. Yet, genome-wide direct Star-PAP targets or the mechanism of specific mRNA recognition is still vague. Here, we employ HITS-CLIP to map the cellular Star-PAP binding landscape and the mechanism of global Star-PAP mRNA association. We show a transcriptome-wide association of Star-PAP that is diminished on Star-PAP depletion. Consistent with its role in the 3′-UTR processing, we observed a high association of Star-PAP at the 3′-UTR region. Strikingly, there is an enrichment of Star-PAP at the coding region exons (CDS) in 42% of target mRNAs. We demonstrate that Star-PAP binding de-stabilises these mRNAs indicating a new role of Star-PAP in mRNA metabolism. Comparison with earlier microarray data reveals that while UTR-associated transcripts are down-regulated, CDS-associated mRNAs are largely up-regulated on Star-PAP depletion. Strikingly, the knockdown of a Star-PAP coregulator RBM10 resulted in a global loss of Star-PAP association on target mRNAs. Consistently, RBM10 depletion compromises 3′-end processing of a set of Star-PAP target mRNAs, while regulating stability/turnover of a different set of mRNAs. Our results establish a global profile of Star-PAP mRNA association and a novel role of Star-PAP in the mRNA metabolism that requires RBM10-mRNA association in the cell.

Regulation of the poly(A) Polymerase Star-PAP by a Nuclear Phosphoinositide Signalosome

“…2A-F ). Star-PAP-PIPKI⍺ complexes were largely nuclear consistent with its role in regulating Star-PAP (Kandala et al , 2019; Koshre et al ., 2021; Laishram, 2018; Mellman et al ., 2008; Sudheesh et al ., 2019), while each of the other Star-PAP complexes were present in the both the nucleus and cytosol. To further establish these interactions, PITP⍺/β, PI4KIIα, IPMK, and PTEN co-IPed with Star-PAP, and their interaction was enhanced by etoposide and DEM ( Fig.…”

“…Under some conditions Star-PAP also exhibits terminal uridylyl transferase (TUT1) activity (Trippe et al, 2006); however, its preference for ATP over UTP indicates that Star-PAP is predominantly a poly (A) polymerase (PAP) . Notably, Star-PAP binds the PIP 5-kinase PIPKI⍺ (PIP5K1A) and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which regulates its PAP activity and controls expression of Star-PAP target mRNAs (Barlow et al, 2010;Mellman et al, 2008)., including HO-1 (Heme oxygenase-1), BIK (Bcl-2-Interacting Killer), cadherin 1, and cadherin 13 (A P & Laishram, 2018;Koshre et al, 2021;Laishram et al, 2011;Li & Anderson, 2014;Li et al, 2013;Li et al, 2012;Li et al, 2017;Mellman & Anderson, 2009;Mellman et al, 2008). Oxidative stress induces Star-PAP phosphorylation by casein kinase I (CKI), which is critical for its PAP activity and priming required for PI(4,5)P2 stimulation, leading to HO-1 mRNA processing (Laishram et al, 2011).…”