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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 (PIPKIα, PIPKIβ and PIPKIγ)1. In addition to functions in the cytosol, phosphoinositides are present in the nucleus2,3, where they modulate several functions4,5,6; 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 pathways1,7. Therefore, to determine roles for nuclear PtdIns4,5P2 we set out to identify proteins that interacted with the nuclear PIPK, PIPKIα. Here we show that PIPKIα 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 PIPKIα regulated-poly(A) polymerase) and that the activity of Star-PAP can be specifically regulated by PtdIns4,5P2. Star-PAP and PIPKIα 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.

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Figure 1: Identification of a nuclear-localized PIPKIα-interacting protein.
Figure 2: Star-PAP has poly(A) polymerase activity that is stimulated by PtdIns4,5P2.
Figure 3: Star-PAP-interacting proteins and identification of Star-PAP target mRNAs.
Figure 4: Star-PAP and PIPKIα are required for efficient 3′ processing of HO-1 mRNA by a mechanism that assembles a Star-PAP complex.

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Primary accessions

Gene Expression Omnibus

Data deposits

The Star-PAP sequence is deposited in the NCBI Library under accession number NP_073741. The microarray data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE9361.

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Acknowledgements

We thank J. L. Manley for the gift of antibodies against CPSF-73, CstF-64 and PAPα; M. Wickens for the generous gift of the PAPα construct and for advice and discussion; D. Brow, S. Miyamoto, D. Wassarman and R. Tibbetts for reading the manuscript and for comments; and C. Song for technical assistance in the early parts of the project. R.A.A. is supported by grants from the National Institutes of Health (NIH). M.L.G., D.L.M. and C.S. were supported by the American Heart Association. C.A.B. is supported by the National Research Service Award. D.L.M. and M.L.G. received Research Training Grant support from the NIH.

Author Contributions D.L.M. contributed to Figs 1a, d, f, 2, 3a, b, 4g–k and Supplementary Figs 1, 2, 4–7. M.L.G. contributed to Figs 1a, 3c, f, 4a–f and Supplementary Fig. 1. C.A.B. contributed to Fig. 3d, e, Supplementary Fig. 1 and Supplementary Tables 1 and 2. C.S. contributed to Fig. 1a, c, e and Supplementary Figs 3 and 6. P.W. and C.K. analysed the microarray data. R.A.A. directed the experimental approach and project. D.L.M., M.L.G., C.A.B. and R.A.A. analysed and interpreted the experiments, and conceptualized and wrote the paper.

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Correspondence to Richard A. Anderson.

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The file contains Supplementary Methods, Supplementary Figures 1-7 with Legends and Supplementary Tables 1-2. (PDF 11680 kb)

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Mellman, D., Gonzales, M., Song, C. et al. A PtdIns4,5P2-regulated nuclear poly(A) polymerase controls expression of select mRNAs. Nature 451, 1013–1017 (2008). https://doi.org/10.1038/nature06666

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