Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co‐transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA‐binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted β‐propeller‐forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C‐terminal domain (CTD) of pol II in vitro and in a two‐hybrid test in vivo. Furthermore, transcriptional run‐on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3′‐end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.

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

[2]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[3]  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.

[4]  Temple F. Smith,et al.  The WD repeat: a common architecture for diverse functions. , 1999, Trends in biochemical sciences.

[5]  E. Cho,et al.  mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. , 1997, Genes & development.

[6]  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.

[7]  B. Séraphin,et al.  Partial purification of the yeast U2 snRNP reveals a novel yeast pre‐mRNA splicing factor required for pre‐spliceosome assembly , 1999, The EMBO journal.

[8]  C. Moore,et al.  Kin28, the TFIIH-Associated Carboxy-Terminal Domain Kinase, Facilitates the Recruitment of mRNA Processing Machinery to RNA Polymerase II , 2000, Molecular and Cellular Biology.

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

[10]  D. Bentley,et al.  5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II. , 1997, Genes & development.

[11]  G. Knapp.  Enzymatic approaches to probing of RNA secondary and tertiary structure. , 1989, Methods in enzymology.

[12]  Aaron J. Shatkin,et al.  The ends of the affair: Capping and polyadenylation , 2000, Nature Structural Biology.

[13]  J. Wootton,et al.  A 127 kDa component of a UV-damaged DNA-binding complex, which is defective in some xeroderma pigmentosum group E patients, is homologous to a slime mold protein. , 1993, Nucleic acids research.

[14]  J. Manley,et al.  RNA polymerase II is an essential mRNA polyadenylation factor , 1998, Nature.

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

[16]  R. Parker,et al.  Effects of mutations in the Saccharomyces cerevisiae RNA14, RNA15, and PAP1 genes on polyadenylation in vivo , 1995, Molecular and cellular biology.

[17]  W. Keller,et al.  Distinct roles of two Yth1p domains in 3′‐end cleavage and polyadenylation of yeast pre‐mRNAs , 2000, The EMBO journal.

[18]  A. Lustig,et al.  Mammalian poly(A)-binding protein II. Physical properties and binding to polynucleotides. , 1993, The Journal of biological chemistry.

[19]  C R Cantor,et al.  Genomic detection of new yeast pre-mRNA 3'-end-processing signals. , 1999, Nucleic acids research.

[20]  J. Manley,et al.  RNA polymerase II and the integration of nuclear events. , 2000, Genes & development.

[21]  A. Yuryev,et al.  The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[22]  N. Proudfoot,et al.  Transcriptional termination signals for RNA polymerase II in fission yeast , 1997, The EMBO journal.

[23]  J. van Helden,et al.  Statistical analysis of yeast genomic downstream sequences reveals putative polyadenylation signals. , 2000, Nucleic acids research.

[24]  N. Proudfoot.  How RNA polymerase II terminates transcription in higher eukaryotes. , 1989, Trends in biochemical sciences.

[25]  P. Silver,et al.  Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation. , 1999, RNA.

[26]  R. Reed,et al.  Characterization of a Protein Complex Containing Spliceosomal Proteins SAPs 49, 130, 145, and 155 , 1999, Molecular and Cellular Biology.

[27]  M. Swanson,et al.  Control of cleavage site selection during mRNA 3′ end formation by a yeast hnRNP , 1998, The EMBO journal.

[28]  J. Manley,et al.  Evolutionarily conserved interaction between CstF-64 and PC4 links transcription, polyadenylation, and termination. , 2001, Molecular cell.

[29]  Rodney Rothstein,et al.  Elevated recombination rates in transcriptionally active DNA , 1989, Cell.

[30]  L. Minvielle-Sebastia,et al.  Coupling termination of transcription to messenger RNA maturation in yeast. , 1998, Science.

[31]  H. Martinson,et al.  Mechanism of Poly(A) Signal Transduction to RNA Polymerase II In Vitro , 2001, Molecular and Cellular Biology.

[32]  C. Moore,et al.  Separation of factors required for cleavage and polyadenylation of yeast pre-mRNA , 1992, Molecular and cellular biology.

[33]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

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

[35]  L. Minvielle-Sebastia,et al.  RNA14 and RNA15 proteins as components of a yeast pre-mRNA 3'-end processing factor. , 1994, Science.

[36]  W. Keller,et al.  Recognition of polyadenylation sites in yeast pre‐mRNAs by cleavage and polyadenylation factor , 2001, The EMBO journal.

[37]  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.

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

[39]  S. Woodson,et al.  Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron. , 1999, RNA.

[40]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

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

[42]  N. Proudfoot,et al.  Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[43]  A. F. Neuwald,et al.  PSI-BLAST searches using hidden markov models of structural repeats: prediction of an unusual sliding DNA clamp and of beta-propellers in UV-damaged DNA-binding protein. , 2000, Nucleic acids research.

[44]  H. Domdey,et al.  Dependence of Yeast Pre-mRNA 3′-End Processing on CFT1: A Sequence Homolog of the Mammalian AAUAAA Binding Factor , 1996, Science.

[45]  N. Proudfoot,et al.  Transcriptional termination factors for RNA polymerase II in yeast. , 2001, Molecular cell.

[46]  N. Proudfoot,et al.  EM visualization of transcription by RNA polymerase II: downstream termination requires a poly(A) signal but not transcript cleavage. , 1999, Molecular cell.