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 promoterproximal 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.Pre-mRNA splicing is a two-step transesterification reaction carried out by the spliceosome, a large and dynamic multicomponent RNA-protein complex (52). The first steps in the assembly of the spliceosome on pre-mRNA involve the recognition of the 5Ј and 3Ј ends of each intron (5Ј and 3Ј splice sites) by small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors. Regulation of this process determines splice site usage in alternative pre-mRNA splicing (50). A report that 40 to 60% of human genes are alternatively spliced to produce multiple gene products (26) underscores the importance of understanding splice site recognition and subsequent spliceosome assembly. Although much progress has been made in recent years toward understanding the biochemical activities of many splicing regulators, it has been difficult to establish systems for examining the roles of such regulators on endogenous pre-mRNAs in vivo and the mechanisms by which they are recruited.An important clue to understanding how splicing factors might initially assemble on pre-mRNA is provided by observations that splicing begins and is sometimes completed cotranscriptionally (for a review, see reference 39). For a number of genes, intron removal has been detected in nascent RNAs still tethered to the DNA axis by RNA polymerase II (Pol II) (3,5,42,53,54,56). Evidence that transcription rates and promoter identity influence alternative splice site selection is consistent with a cotranscriptional splicing mechanism in humans (9, 21, 45) and yeast (K. J. Howe, C. M. Kane, and M. Ares, unpublished data). The findings that the C-terminal domain (CTD) of RNA Pol II is required for efficient capping, splicing, and polyadenylation of pre-mRNA (33) and specifically stimulates spl...