Cotranscriptional Recruitment of the U1 snRNP to Intron-Containing Genes in Yeast

Abstract

Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5′ splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated. Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes. In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal. Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP. Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention.

ACKNOWLEDGMENTS

We thank L. Goetsch, B. Seraphin, and R. Iggo for strains; E. Young and W. Zacharaie for the tagging plasmids; and M. Ares for communicating unpublished results. We are grateful to the Gottschling lab for sharing their ChIP protocol, J. Delrow and J. Howard for help with the microarray analysis, and A. Hopper, D. Stanek, F. Stewart, J. Valcarcel, and A. Weiner for discussions and comments on the manuscript.

K.M.K. is the recipient of a predoctoral fellowship from Boehringer Ingelheim Fonds. Supported by the Max Planck Gesellschaft and a Research Project Grant (RPG-00-110-01-MGO) from the American Cancer Society.