Direct Interactions Between the Paf1 Complex and a Cleavage and Polyadenylation Factor Are Revealed by Dissociation of Paf1 from RNA Polymerase II

Department of Biochemistry and Molecular Genetics, and Molecular Biology Program, University of Colorado School of Medicine, 12801 East 17^th Ave, P.O. Box 6511, Aurora, CO 80045; Department of Biology, Regis University, Denver CO, 80221

^* To whom correspondence should be addressed. Email: Judith.Jaehning{at}UCHSC.edu.

	Abstract

The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1 and Leo1) is normally associated with RNA polymerase II (pol II) throughout the transcription cycle. However, loss of either Rtf1 or Cdc73 results in detachment of the Paf1 complex from pol II and the chromatin form of actively transcribed genes. Using functional tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components remain stably associated with each other in an RNAse-resistant complex after dissociation from pol II and chromatin. Loss of Paf1 or Ctr9, or to a lesser extent Cdc73 or Rtf1, results in reduced levels of serine 2 phosphorylation of the pol II C-terminal domain and to increased read-through of the MAK21 polyadenylation site. We find that the cleavage and polyadenylation factor Cft1 requires the pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5 phosphorylated form of pol II. When the Paf1 complex is dissociated from pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3' end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3' end formation observed in the absence of Paf1.