To obtain insight into the function of dPcf11 in Drosophila, immunofluorescence microscopy was used to analyze the distribution on polytene chromosomes of a protein that had 37% amino acid sequence identity with the CTD interacting domain of yPcf11 (Meinhart, 2004). Antibodies were raised against an N-terminal polypeptide encompassing amino acids 1–238. In accordance with one form of dPcf11 predicted from cDNA sequence information, Western blotting detected a single polypeptide of ~60 kDa in whole-cell lysates from Drosophila salivary glands and tissue culture cells (Zhang, 2006).

Immunofluorescence analysis revealed that dPcf11 was concentrated at a small number of loci on polytene chromosomes. For chromosomes from either nonheat-shocked larvae or heat-shocked larvae, all locations staining intensely for dPcf11 also stained intensely for Pol II. The chromosomes from heat-shocked larvae provide the strongest indication that dPcf11 is involved in transcription. Heat shock causes the induction of a small collection of genes whose locations on the chromosomes are known. dPcf11 is concentrated at every one of these locations. In addition to the naturally occurring genes, dPcf11 was also detected on a heat shock-inducible transgene located at 87E (Wu, 2003). Interestingly, dPcf11 was not detected at the myriad of locations on the chromosome from nonheat-shocked larvae that modestly stain with antibody against Pol II. Many of these locations are likely to contain Pol II molecules that have paused as a result of the negative elongation factor Negative elongation factor E (NELF) (Wu, 2003; Wu, 2005; Zhang, 2006 and references therein).

Locus 87A on some polytene chromosomes from heat-shocked larvae exhibited very intriguing patterns of dPcf11 and Pol II staining. 87A contains two copies of the hsp70 gene that are divergently transcribed. When this region was stretched during preparation of the chromosomes, the staining pattern for each protein appeared as two bands. Antibody against Pol II stained two regions toward the center of the locus, whereas antibody against dPcf11 stained two regions toward the periphery. These patterns of staining suggest that dPcf11 is concentrated toward the 3′ ends of each copy of hsp70 (Zhang, 2006).

Reference names in red indicate recommended papers.

Adelman, K., et al. (2005). Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol. Cell 17(1): 103-12. 15629721

Ahn, S.H., Kim, M. and Buratowski, S. (2004). Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3' end processing. Mol. Cell 13(1): 67-76. 14731395

Amrani, N., et al. (1997). PCF11 encodes a third protein component of yeast cleavage and polyadenylation factor I. Mol. Cell. Biol. 17(3): 1102-9. 9032237

Barilla, D., Lee, B. A., and Proudfoot, N. J. (2001). Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 98: 445-450. 11149954

Bentley, D. (2002). The mRNA assembly line: Transcription and processing machines in the same factory. Curr. Opin. Cell Biol. 14: 336-342. 12067656

Birse, C. E., et al. (1998). Coupling termination of transcription to messenger RNA maturation in yeast. Science 280(5361): 298-301. 9535662

Boehm, A. K., Saunders, A., Werner, J. and Lis, J. T. (2003). Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol. Cell. Biol. 23: 7628-7637. 14560008

Buratowski, S. (2003). The CTD code. Nat. Struct. Biol. 10: 679-680. 12942140

Buratowski, S. (2005). Connections between mRNA 3' end processing and transcription termination. Curr. Opin. Cell Biol. 17: 257-261. 15901494

Dantonel, J. C., Murthy, K. G., Manley, J. L, and Tora, L. (1997). Transcription factor TFIID recruits factor CPSF for formation of 3' end of mRNA. Nature 389: 399-402. 9311784

de Vries, H., et al. (2000). Human pre-mRNA cleavage factor II(m) contains homologs of yeast proteins and bridges two other cleavage factors. EMBO J. 19: 5895-5904. 11060040

Gross, S. and Moore C. (2001a). Five subunits are required for reconstitution of the cleavage and polyadenylation activities of Saccharomyces cerevisiae cleavage factor I. Proc. Natl. Acad. Sci. 98: 6080-6085. 11344258

Gross, S. and Moore C. (2001b). Rna15 interaction with the A-rich yeast polyadenylation signal is an essential step in mRNA 3'-end formation. Mol. Cell. Biol. 21: 8045-8055. 11689695

Hollingworth, D., Noble, C. G., Taylor, I. A. and Ramos A. (2006). RNA polymerase II CTD phosphopeptides compete with RNA for the interaction with Pcf11. RNA 12(4): 555-60. 16497660

Kim, M., Ahn, S. H., Krogan, N. J., Greenblatt, J. F., and Buratowski, S. (2004a). Transitions in RNA polymerase II elongation complexes at the 3' ends of genes. EMBO J. 23: 354-364. 14739930

Kim, M., Krogan, N. J., Vasiljeva, L., Rando, O. J., Nedea, E., Greenblatt, J. F., and Buratowski, S. (2004b). The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432: 517-522. 15565157

Kireeva, M. L., Komissarova, N., Waugh, D. S., and Kashlev, M. (2000). The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex. J. Biol. Chem. 275: 6530-6536. 10692458

Komissarova, N., Becker, J., Solter, S., Kireeva, M., and Kashlev, M. (2002). Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination. Mol. Cell 10: 1151-1162. 12453422

Krishnamurthy S, He X, Reyes-Reyes M, Moore C, Hampsey M. (2004). Ssu72 is an RNA polymerase II CTD phosphatase. Mol. Cell 14: 387-394. 15125841

Lang, W. H., Platt, T. and Reeder, R. H. (1998). Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III. Proc. Natl. Acad. Sci. 95: 4900-4905. 9560200

Licatalosi, D. D., Geiger, G., Minet, M., Schroeder, S., Cilli, K., McNeil, J. B. and Bentley. D. L. (2002). Functional interaction of yeast pre-mRNA 3' end processing factors with RNA polymerase II. Mol. Cell 9: 1101-1111. 12049745

Luo, W. and Bentley, D. (2004). A ribonucleolytic rat torpedoes RNA polymerase II. Cell 119: 911-914. 15620350

Luo, W., Johnson, A. W. and Bentley, D. L. (2006). The role of Rat1 in coupling mRNA 3'-end processing to transcription termination: implications for a unified allosteric-torpedo model. Genes Dev. 20(8): 954-65. 16598041

McCracken, S., Fong, N., Yankulov, K., Ballantyne, S., Pan, G., Greenblatt, J., Patterson, S. D., Wickens, M. and Bentley, D. L. (1997). The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385: 357-361. 9002523

Meinhart, A. and Cramer, P. (2004). Recognition of RNA polymerase II carboxy-terminal domain by 3'-RNA-processing factors. Nature 430: 223-226. 15241417

Noble, C. G., Hollingworth, D., Martin, S. R., Ennis-Adeniran, V., Smerdon, S. J., Kelly, G., Taylor, I. A. and Ramos, A. (2005). Key features of the interaction between Pcf11 CID and RNA polymerase II CTD. Nat. Struct. Mol. Biol. 12: 144-151. 15665873

Park, N. J., Tsao, D. C., and Martinson, H. G. (2004). The two steps of poly(A)-dependent termination, pausing and release, can be uncoupled by truncation of the RNA polymerase II carboxyl-terminal repeat domain. Mol. Cell. Biol. 24: 4092-4103. 15121832

Plant, K. E. et al. (2005). Strong polyadenylation and weak pausing combine to cause efficient termination of transcription in the human Ggamma-globin gene. Mol. Cell. Biol. 25: 3276-3285. 15798211

Price, D. H. (2000). P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol. Cell. Biol. 20: 2629-2634. 10733565

Proudfoot, N. J. (2004). New perspectives on connecting messenger RNA 3' end formation to transcription. Curr. Opin. Cell Biol. 16: 272-278. 15145351

Proudfoot, N. J., Furger, A. and Dye, M. J. (2002). Integrating mRNA processing with transcription. Cell 108: 501-512. 11909521

Renner, D.B., Yamaguchi, Y., Wada, T., Handa, H., and Price, D.H. (2201). A highly purified RNA polymerase II elongation control system. J. Biol. Chem. 276: 42601-42609. 11553615

Richardson, J.P. (2002). Rho-dependent termination and ATPases in transcript termination. Biochim. Biophys. Acta 1577: 251-260. 11553615

Sadowski, M., Dichtl, B., Hubner, W. and Keller, W. (2003). Independent functions of yeast Pcf11p in pre-mRNA 3' end processing and in transcription termination. EMBO J. 22: 2167-2177. 12727883

Steinmetz, E. J. and Brow, D. A. (2003). Ssu72 protein mediates both poly(A)-coupled and poly(A)-independent termination of RNA polymerase II transcription. Mol. Cell. Biol. 23: 6339-6349. 12944462

Toulokhonov, I. and Landick, R. (2003). The flap domain is required for pause RNA hairpin inhibition of catalysis by RNA polymerase and can modulate intrinsic termination, Mol. Cell 12: 1125-1136. 14636572

Venkataraman, K., Venkataraman, K., Brown, K. M. and Gilmartin, G. M. (2005). Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition. Genes Dev. 19: 1315-1327. 15937220

Wada, T., Takagi, T., Yamaguchi, Y., Ferdous, A., Imai, T., Hirose, S., Sugimoto, S., Yano, K., Hartzog, G.A. and Winston, F. (1998). DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. Genes & Dev. 12: 343-356. 9450929

West, S., Gromak, N. and Proudfoot, N. J. (2004). Human 5' ? 3' exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature 432: 522-525. 15565158

Wu, C. H., et al. (2003). . NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila. Genes Dev. 17: 1402-1414. 12782658

Wu, C. H., et al. (2005). Molecular characterization of Drosophila NELF. Nucleic Acids Res. 33: 1269-1279. 15741180

Yamaguchi, Y., Takagi, T., Wada, T., Yano, K., Furuya, A., Sugimoto, S., Hasegawa, J. and Handa, H. (1999). NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97: 41-51. 10199401

Yamaguchi, Y., Inukai, N., Narita, T., Wada, T. and Handa, H. (2002). Evidence that Negative Elongation Factor represses transcription elongation through binding to a DRB sensitivity-inducing factor/RNA polymerase II complex and RNA. Mol. Cell. Biol. 22: 2918-2927. 11940650

Zhang, Z., Wu, C. H. and Gilmour, D. S. (2004). Analysis of polymerase II elongation complexes by native gel electrophoresis. Evidence for a novel carboxyl-terminal domain-mediated termination mechanism. J. Biol. Chem. 279(22): 23223-8. 15056674

Zhang, Z., Fu, J. and Gilmour, D. S. (2005). CTD-dependent dismantling of the RNA polymerase II elongation complex by the pre-mRNA 3'-end processing factor, Pcf11. Genes Dev. 19(13): 1572-80. 15998810

Zhang, Z. and Gilmour, D. S. (2006). Pcf11 is a termination factor in Drosophila that dismantles the elongation complex by bridging the CTD of RNA polymerase II to the nascent transcript. Mol. Cell 21(1): 65-74. 16387654

Zhao, J., Hyman, L. and Moore. C. (1999). Formation of mRNA 3' ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis, Microbiol. Mol. Biol. Rev. 63: 405-445. 10357856

date revised: 10/12/2006

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