CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3′ processing

AAUAAA is the most highly conserved motif in eukaryotic mRNA polyadenylation sites and, in mammals, is specifically recognized by the multisubunit CPSF (cleavage and polyadenylation specificity factor) complex. Despite its critical functions in mRNA 3' end formation, the molecular basis for CPSF-AAUAAA interaction remains poorly defined. The CPSF subunit CPSF160 has been implicated in AAUAAA recognition, but direct evidence has been lacking. Using in vitro and in vivo assays, we unexpectedly found that CPSF subunits CPSF30 and Wdr33 directly contact AAUAAA. Importantly, the CPSF30-RNA interaction is essential for mRNA 3' processing and is primarily mediated by its zinc fingers 2 and 3, which are specifically targeted by the influenza protein NS1A to suppress host mRNA 3' processing. Our data suggest that AAUAAA recognition in mammalian mRNA 3' processing is more complex than previously thought and involves multiple protein-RNA interactions.

[1]  G. Christofori,et al.  3′ cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP , 1988, Cell.

[2]  Jing Zhao,et al.  Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis , 1999, Microbiology and Molecular Biology Reviews.

[3]  B. Tian,et al.  Alternative cleavage and polyadenylation: the long and short of it. , 2013, Trends in biochemical sciences.

[4]  Christopher R. Sibley,et al.  iCLIP: Protein–RNA interactions at nucleotide resolution , 2014, Methods.

[5]  R. Russell,et al.  WD40 proteins propel cellular networks. , 2010, Trends in biochemical sciences.

[6]  M. Hentze,et al.  3′ end mRNA processing: molecular mechanisms and implications for health and disease , 2008, The EMBO journal.

[7]  T. Toda,et al.  Inactivation of the Pre-mRNA Cleavage and Polyadenylation Factor Pfs2 in Fission Yeast Causes Lethal Cell Cycle Defects , 2005, Molecular and Cellular Biology.

[8]  R. Krug,et al.  Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs. , 1998, Molecular cell.

[9]  Scott B. Dewell,et al.  Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP , 2010, Cell.

[10]  J. Yates,et al.  Molecular architecture of the human pre-mRNA 3' processing complex. , 2009, Molecular cell.

[11]  M. Rosbash,et al.  The U1 snRNP protein U1C recognizes the 5′ splice site in the absence of base pairing , 2002, Nature.

[12]  L. Minvielle-Sebastia,et al.  The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins. , 1997, Genes & development.

[13]  L. Tong,et al.  Protein factors in pre-mRNA 3′-end processing , 2008, Cellular and Molecular Life Sciences.

[14]  N. Proudfoot.  Ending the message: poly(A) signals then and now. , 2011, Genes & development.

[15]  B. Graveley,et al.  CPSF recognition of an HIV-1 mRNA 3'-processing enhancer: multiple sequence contacts involved in poly(A) site definition. , 1995, Genes & development.

[16]  K. Nishida,et al.  Mechanisms and consequences of alternative polyadenylation. , 2011, Molecules and Cells.

[17]  Diana Blank,et al.  Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination , 2002, The EMBO journal.

[18]  S. Vagner,et al.  Molecular mechanisms of eukaryotic pre-mRNA 3′ end processing regulation , 2009, Nucleic acids research.

[19]  Q. Li,et al.  Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit1[OA] , 2006, Plant Physiology.

[20]  Guoli Ji,et al.  Genome-Wide Control of Polyadenylation Site Choice by CPSF30 in Arabidopsis[C][W][OA] , 2012, Plant Cell.

[21]  M. Wickens,et al.  Point mutations in AAUAAA and the poly (A) addition site: effects on the accuracy and efficiency of cleavage and polyadenylation in vitro. , 1990, Nucleic acids research.

[22]  L. Tong,et al.  Delineating the Structural Blueprint of the Pre-mRNA 3′-End Processing Machinery , 2014, Molecular and Cellular Biology.

[23]  V. Quesada,et al.  FY Is an RNA 3′ End-Processing Factor that Interacts with FCA to Control the Arabidopsis Floral Transition , 2003, Cell.

[24]  Patrick Schultz,et al.  Recent advances in understanding the structure and function of general transcription factor TFIID , 2009, Cellular and Molecular Life Sciences.

[25]  M. Moore,et al.  The human Prp8 protein is a component of both U2- and U12-dependent spliceosomes. , 1999, RNA.

[26]  E. Fleming,et al.  Activation of HIV‐1 pre‐mRNA 3′ processing in vitro requires both an upstream element and TAR. , 1992, The EMBO journal.

[27]  Yongsheng Shi,et al.  Alternative polyadenylation: new insights from global analyses. , 2012, RNA.

[28]  Y. Qi,et al.  Nuclear pre-mRNA 3′-end processing regulates synapse and axon development in C. elegans , 2010, Development.

[29]  M. Zavolan,et al.  Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33 , 2014, Genes & development.

[30]  W. Marzluff,et al.  A core complex of CPSF73, CPSF100, and Symplekin may form two different cleavage factors for processing of poly(A) and histone mRNAs. , 2009, Molecular cell.

[31]  Jennifer L. Bachorik,et al.  Gemin5-snRNA interaction reveals an RNA binding function for WD repeat domains , 2009, Nature Structural &Molecular Biology.

[32]  K. Murthy,et al.  The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation. , 1995, Genes & development.

[33]  J. Manley,et al.  Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability. , 1996, Genes & development.

[34]  J. Manley,et al.  Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation , 1988, Cell.

[35]  J. Manley,et al.  Mechanism and regulation of mRNA polyadenylation. , 1997, Genes & development.

[36]  K. Murthy,et al.  Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus. , 1992, The Journal of biological chemistry.

[37]  G. Christofori,et al.  Cleavage and polyadenylation factor CPF specifically interacts with the pre‐mRNA 3′ processing signal AAUAAA. , 1991, The EMBO journal.

[38]  Mihaela Zavolan,et al.  Genome-wide analysis of pre-mRNA 3' end processing reveals a decisive role of human cleavage factor I in the regulation of 3' UTR length. , 2012, Cell reports.

[39]  W. Keller,et al.  The WD‐repeat protein Pfs2p bridges two essential factors within the yeast pre‐mRNA 3′‐end‐processing complex , 2000, The EMBO journal.

[40]  Rei-Lin Kuo,et al.  The CPSF30 Binding Site on the NS1A Protein of Influenza A Virus Is a Potential Antiviral Target , 2006, Journal of Virology.

[41]  Ji Wan,et al.  Transcriptome-wide analyses of CstF64–RNA interactions in global regulation of mRNA alternative polyadenylation , 2012, Proceedings of the National Academy of Sciences.

[42]  J. Alwine,et al.  Functionally Significant Secondary Structure of the Simian Virus 40 Late Polyadenylation Signal , 2000, Molecular and Cellular Biology.

[43]  W. Keller,et al.  Human Fip1 is a subunit of CPSF that binds to U‐rich RNA elements and stimulates poly(A) polymerase , 2004, The EMBO journal.

[44]  Xiaohui S. Xie,et al.  Fip1 regulates mRNA alternative polyadenylation to promote stem cell self‐renewal , 2014, The EMBO journal.

[45]  E. Wahle,et al.  Purification of the cleavage and polyadenylation factor involved in the 3'-processing of messenger RNA precursors. , 1991, The Journal of biological chemistry.

[46]  Yongsheng Shi,et al.  Pre‐mRNA 3′‐end processing complex assembly and function , 2011, Wiley interdisciplinary reviews. RNA.

[47]  Jeroen Krijgsveld,et al.  The RNA-binding protein repertoire of embryonic stem cells , 2013, Nature Structural &Molecular Biology.

[48]  Norman E. Davey,et al.  Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins , 2012, Cell.