Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition.

At least half of all human pre-mRNAs are subject to alternative 3' processing that may modulate both the coding capacity of the message and the array of post-transcriptional regulatory elements embedded within the 3' UTR. Vertebrate poly(A) site selection appears to rely primarily on the binding of CPSF to an A(A/U)UAAA hexamer upstream of the cleavage site and CstF to a downstream GU-rich element. At least one-quarter of all human poly(A) sites, however, lack the A(A/U)UAAA motif. We report that sequence-specific RNA binding of the human 3' processing factor CFI(m) can function as a primary determinant of poly(A) site recognition in the absence of the A(A/U)UAAA motif. CFI(m) is sufficient to direct sequence-specific, A(A/U)UAAA-independent poly(A) addition in vitro through the recruitment of the CPSF subunit hFip1 and poly(A) polymerase to the RNA substrate. ChIP analysis indicates that CFI(m) is recruited to the transcription unit, along with CPSF and CstF, during the initial stages of transcription, supporting a direct role for CFI(m) in poly(A) site recognition. The recognition of three distinct sequence elements by CFI(m), CPSF, and CstF suggests that vertebrate poly(A) site definition is mechanistically more similar to that of yeast and plants than anticipated.

[1]  W. Keller,et al.  Purification and Characterization of Human Cleavage Factor I Involved in the 3′ End Processing of Messenger RNA Precursors (*) , 1996, The Journal of Biological Chemistry.

[2]  Anton J. Enright,et al.  Human MicroRNA Targets , 2004, PLoS biology.

[3]  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.

[4]  E. Wahle,et al.  Poly(A) Tail Length Control Is Caused by Termination of Processive Synthesis (*) , 1995, The Journal of Biological Chemistry.

[5]  J. Nevins,et al.  Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA , 1991, Molecular and cellular biology.

[6]  M. Wickens,et al.  Polyadenylation of mRNA: minimal substrates and a requirement for the 2' hydroxyl of the U in AAUAAA , 1990, Molecular and cellular biology.

[7]  W. Keller,et al.  Human pre-mRNA cleavage factor Im is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits. , 1998, Molecular cell.

[8]  D. Gautheret,et al.  Patterns of variant polyadenylation signal usage in human genes. , 2000, Genome research.

[9]  I. Mattaj,et al.  Involvement of the carboxyl terminus of vertebrate poly(A) polymerase in U1A autoregulation and in the coupling of splicing and polyadenylation. , 1997, Genes & development.

[10]  M. Wickens,et al.  The C-terminal domain of RNA polymerase II couples mRNA processing to transcription , 1997, Nature.

[11]  E. Wahle,et al.  Assembly of a processive messenger RNA polyadenylation complex. , 1993, The EMBO journal.

[12]  A. Furger,et al.  Integrating mRNA Processing with Transcription , 2002, Cell.

[13]  Richard C. Moore,et al.  The early stages of duplicate gene evolution , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Kirk M Brown,et al.  A mechanism for the regulation of pre-mRNA 3' processing by human cleavage factor Im. , 2003, Molecular cell.

[15]  Bom Ko,et al.  Identification of new poly(A) polymerase-inhibitory proteins capable of regulating pre-mRNA polyadenylation. , 2002, Journal of molecular biology.

[16]  Jurg Ott,et al.  Nucleotide frequency variation across human genes. , 2003, Genome research.

[17]  C R Cantor,et al.  In silico detection of control signals: mRNA 3'-end-processing sequences in diverse species. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[18]  S. Buratowski,et al.  Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. , 2004, Molecular cell.

[19]  François-Michel Boisvert,et al.  A Proteomic Analysis of Arginine-methylated Protein Complexes* , 2003, Molecular & Cellular Proteomics.

[20]  E. Cho,et al.  Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. , 2000, Genes & development.

[21]  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.

[22]  Nick Proudfoot,et al.  Polyadenylation: A tail of two complexes , 2002, Current Biology.

[23]  J. Alwine,et al.  The human immunodeficiency virus type 1 polyadenylylation signal: a 3' long terminal repeat element upstream of the AAUAAA necessary for efficient polyadenylylation. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Manley,et al.  Identification and Functional Characterization of Neo-Poly(A) Polymerase, an RNA Processing Enzyme Overexpressed in Human Tumors , 2022 .

[25]  D. Licatalosi,et al.  Functional interaction of yeast pre-mRNA 3' end processing factors with RNA polymerase II. , 2002, Molecular cell.

[26]  Nick Proudfoot,et al.  New perspectives on connecting messenger RNA 3' end formation to transcription. , 2004, Current opinion in cell biology.

[27]  W. Keller,et al.  Last but Not Least Regulated Poly(A) Tail Formation , 1999, Cell.

[28]  Y. Lee,et al.  Interaction of poly(A) polymerase with the 25-kDa subunit of cleavage factor I. , 2001, Biochemical and biophysical research communications.

[29]  D. Bentley,et al.  The link between mRNA processing and transcription: communication works both ways. , 2004, Experimental cell research.

[30]  P. Silver,et al.  Intron status and 3'-end formation control cotranscriptional export of mRNA. , 2002, Genes & development.

[31]  I. Mattaj,et al.  U1 snRNP inhibits pre-mRNA polyadenylation through a direct interaction between U1 70K and poly(A) polymerase. , 1998, Molecular cell.

[32]  N. Proudfoot,et al.  Recruitment of a Basal Polyadenylation Factor by the Upstream Sequence Element of the Human Lamin B2 Polyadenylation Signal , 2000, Molecular and Cellular Biology.

[33]  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.

[34]  J. Alwine,et al.  Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses , 1992, Molecular and cellular biology.

[35]  P. Silver,et al.  Arginine methyltransferase affects interactions and recruitment of mRNA processing and export factors. , 2004, Genes & development.

[36]  A. Virtanen,et al.  A Novel Nuclear Human Poly(A) Polymerase (PAP), PAPγ* , 2001, The Journal of Biological Chemistry.

[37]  C. Prives,et al.  Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non‐consensus sites , 1998, The EMBO journal.

[38]  E. Wahle,et al.  Structure and function of poly(A) binding proteins. , 2004, Biochimica et biophysica acta.

[39]  B. Graveley.  Sorting out the complexity of SR protein functions. , 2000, RNA.

[40]  I. Mattaj,et al.  The carboxyl terminus of vertebrate poly(A) polymerase interacts with U2AF 65 to couple 3'-end processing and splicing. , 2000, Genes & development.

[41]  N. Krogan,et al.  Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes , 2004, The EMBO journal.

[42]  J. Dantonel,et al.  Transcription factor TFIID recruits factor CPSF for formation of 3′ end of mRNA , 1997, Nature.

[43]  P. Sharp,et al.  RNA Polymerase II Accumulation in the Promoter-Proximal Region of the Dihydrofolate Reductase and γ-Actin Genes , 2003, Molecular and Cellular Biology.

[44]  P. Silver,et al.  Arginine methylation facilitates the nuclear export of hnRNP proteins. , 1998, Genes & development.

[45]  C. V. Jongeneel,et al.  Long-range heterogeneity at the 3' ends of human mRNAs. , 2002, Genome research.

[46]  C. Murphy,et al.  Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes. , 1999, Molecular biology of the cell.

[47]  S. Cardinale,et al.  Distinct Sequence Motifs within the 68-kDa Subunit of Cleavage Factor Im Mediate RNA Binding, Protein-Protein Interactions, and Subcellular Localization* , 2004, Journal of Biological Chemistry.

[48]  R. Russnak.  Regulation of polyadenylation in hepatitis B viruses: stimulation by the upstream activating signal PS1 is orientation-dependent, distance-independent, and additive. , 1991, Nucleic acids research.

[49]  M. Mann,et al.  Large-scale Proteomic Analysis of the Human Spliceosome References , 2006 .

[50]  J. Manley,et al.  Strange bedfellows: polyadenylation factors at the promoter. , 2003, Genes & development.

[51]  A. Scorilas,et al.  Polyadenylate polymerase enzymatic activity in mammary tumor cytosols: A new independent prognostic marker in primary breast cancer. , 2000, Cancer research.

[52]  Y. Aissouni,et al.  The Cleavage/Polyadenylation Activity Triggered by a U-rich Motif Sequence Is Differently Required Depending on the Poly(A) Site Location at Either the First or Last 3′-Terminal Exon of the 2′-5′ Oligo(A) Synthetase Gene* , 2002, The Journal of Biological Chemistry.

[53]  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.

[54]  G. Edwalds-Gilbert,et al.  Alternative poly(A) site selection in complex transcription units: means to an end? , 1997, Nucleic acids research.

[55]  Jeffrey Wilusz,et al.  Upstream Elements Present in the 3′-Untranslated Region of Collagen Genes Influence the Processing Efficiency of Overlapping Polyadenylation Signals* , 2002, The Journal of Biological Chemistry.

[56]  C. Prives,et al.  Cell-cycle related regulation of poly(A) polymerase by phosphorylation , 1996, Nature.

[57]  J. Manley,et al.  Deregulation of Poly(A) Polymerase Interferes with Cell Growth , 1998, Molecular and Cellular Biology.

[58]  Sita Awasthi,et al.  Association of polyadenylation cleavage factor I with U1 snRNP. , 2003, RNA.

[59]  Hana Kim,et al.  Regulation of poly(A) polymerase by 14‐3‐3ϵ , 2003, EMBO Journal.

[60]  C. Moore,et al.  Rna15 Interaction with the A-Rich Yeast Polyadenylation Signal Is an Essential Step in mRNA 3′-End Formation , 2001, Molecular and Cellular Biology.

[61]  Daniel Zenklusen,et al.  Coupling of Termination, 3′ Processing, and mRNA Export , 2002, Molecular and Cellular Biology.

[62]  J. Manley,et al.  The Polyadenylation Factor CstF-64 Regulates Alternative Processing of IgM Heavy Chain Pre-mRNA during B Cell Differentiation , 1996, Cell.

[63]  Steven P. Gygi,et al.  Comprehensive proteomic analysis of the human spliceosome , 2002, Nature.