A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs

Abstract

Phosphoinositides are a family of lipid signalling molecules that regulate many cellular functions in eukaryotes. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2), the central component in the phosphoinositide signalling circuitry, is generated primarily by type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIalpha, PIPKIbeta and PIPKIgamma). In addition to functions in the cytosol, phosphoinositides are present in the nucleus, where they modulate several functions; however, the mechanism by which they directly regulate nuclear functions remains unknown. PIPKIs regulate cellular functions through interactions with protein partners, often PtdIns4,5P2 effectors, that target PIPKIs to discrete subcellular compartments, resulting in the spatial and temporal generation of PtdIns4,5P2 required for the regulation of specific signalling pathways. Therefore, to determine roles for nuclear PtdIns4,5P2 we set out to identify proteins that interacted with the nuclear PIPK, PIPKIalpha. Here we show that PIPKIalpha co-localizes at nuclear speckles and interacts with a newly identified non-canonical poly(A) polymerase, which we have termed Star-PAP (nuclear speckle targeted PIPKIalpha regulated-poly(A) polymerase) and that the activity of Star-PAP can be specifically regulated by PtdIns4,5P2. Star-PAP and PIPKIalpha function together in a complex to control the expression of select mRNAs, including the transcript encoding the key cytoprotective enzyme haem oxygenase-1 (refs 8, 9) and other oxidative stress response genes by regulating the 3'-end formation of their mRNAs. Taken together, the data demonstrate a model by which phosphoinositide signalling works in tandem with complement pathways to regulate the activity of Star-PAP and the subsequent biosynthesis of its target mRNA. The results reveal a mechanism for the integration of nuclear phosphoinositide signals and a method for regulating gene expression.

Inositol 1,4,5-Triphosphate Receptor-binding Protein Released with Inositol 1,4,5-Triphosphate (IRBIT) Associates with Components of the mRNA 3′ Processing Machinery in a Phosphorylation-dependent Manner and Inhibits Polyadenylation

“…Alterations of the cellular environment such as oxidative stress lead to induced transcription of cytoprotective genes and consequently to accumulation of the corresponding transcripts and enhanced translation (19). Recent evidence suggests that the cell has evolved specific mechanisms for 3Ј processing of antioxidant response mRNAs (15). Given that protein synthesis represents one of the most energy-consuming functions, temporary attenuation of polyadenylation by IRBIT may be a way to silence the translation of nonessential transcripts and to limit the energetic cost of antioxidant response, therefore promoting long term survival of the cell.…”

“…Enhanced 3Ј processing of a specific population of pre-mRNAs in response to oxidative stress mediated by tBHQ has been reported recently (15). We first investigated whether this stimulus-dependent alteration of 3Ј RNA processing is associated with changes in the phosphorylation state of IRBIT.…”

“…The findings that in vitro, the CPSF subunit Fip1 interacts with both PAP and RNA and also stimulates PAP activity led to the hypothesis that Fip1 recruits PAP to RNA, therefore contributing to the assembly of the 3Ј processing complex (11). In addition to the constitutive cleavage and polyadenylation of all mRNAs, stress-dependent control of 3Ј processing emerged recently as a novel mechanism to regulate gene expression in response to a stimulus (15)(16)(17)(18). However, the involvement of CPSF and PAP to stress-responsive cleavage and polyadenylation remains largely unexplored.…”

CKIα Is Associated with and Phosphorylates Star-PAP and Is Also Required for Expression of Select Star-PAP Target Messenger RNAs

“…This is often achieved through specific interactions between the phosphoinositide-generating enzymes and targeting factors, which are themselves often PI-4,5-P 2 -regulated proteins (13,14). This allows for the generation of inositol lipid second messengers at specific sites within the cell that, in turn, allows for precise targeting of individual phosphoinositide-sensitive pathways (12).In the nucleus, PI-4,5-P 2 and PIPKI␣, as well as Star-PAP, are found in structures called nuclear speckles (6,15), which are nuclear bodies enriched in factors required for the processing of pre-mRNA (16). The direct interaction between Star-PAP and PIPKI␣ and the proximity of Star-PAP to PI-4,5-P 2 generation in nuclear speckles suggest that nuclear phosphoinositides are spatially positioned to modulate Star-PAP activity in vivo.…”

PtdIns(4,5)P 2 signaling regulates ATG14 and autophagy

“…The majority of PtdIns(4,5)P 2 in mammalian cells are generated by the type I phosphatidylinositol 4-phosphate (PIP) 5-kinase (PIPKI), of which there are three genes (α, β, and γ) each with multiple splicing isoforms (11). PIPKIα controls a nuclear PtdIns(4,5)P 2 pathway that regulates the mRNA processing of select genes activated by different cellular stresses (12). PIPKIγi2 partially targets recycling endosomes and promotes the recycling of integrins and epithelial cadherin by generating PtdIns(4,5)P 2 and regulating exocyst and adaptor protein complexes (13)(14)(15), whereas PIPKIγi5 is found at endosomal compartments where it mediates PtdIns(4,5)P 2 signaling to regulate the endolysosomal trafficking of epidermal growth factor receptor (EGFR) (16,17).…”