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Dual Roles for Spt5 in Pre-mRNA Processing and Transcription Elongation Revealed by Identification of Spt5-Associated Proteins

D. L. Lindstrom,¹ S. L. Squazzo,¹ N. Muster,² T. A. Burckin,¹ K. C. Wachter,^1, C. A. Emigh,¹ J. A. McCleery,¹ J. R. Yates III,² and G. A. Hartzog^1*

Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064,¹ Department of Cell Biology, Scripps Research Institute, La Jolla, California 92130²

Received 16 September 2002/ Returned for modification 22 October 2002/ Accepted 14 November 2002

During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.

Present address: Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195.

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