The Rpb4 subunit of RNA polymerase II contributes to cotranscriptional recruitment of 3' processing factors.

The synthesis of eukaryotic mRNA by RNA polymerase II (RNApII) is a multistep process involving initiation, elongation, and termination. During the transcription cycle, RNApII associates with many proteins involved in the regulation of these processes, including the basal transcription factors, coactivators, elongation factors, and factors involved in 3′-end formation and termination (6). This regulation of transcription by RNApII and its associated proteins is critical for gene expression. The RNApII enzyme from the yeast Saccharomyces cerevisiae is a complex of 12 subunits, Rpb1 to Rpb12, that can dissociate into a 10-subunit core and a heterodimer consisting of Rpb4 and Rpb7 (6). Rpb4 is not essential during optimal growth conditions, although the deletion strain grows very slowly (32). RNApII purified from an rpb4Δ strain lacks Rpb4 but also contains no detectable level of Rpb7 (8). It has been shown that Rpb7, a subunit that is essential for cell viability (22), can interact with polymerase independently of Rpb4, but this interaction is weak and can easily be detected only when Rpb7 is overexpressed (28). RNApII lacking Rpb4/7 is catalytically active for polymerization, but the heterodimer is required for promoter-dependent transcription in vitro (8). Crystal structures show the location of the Rpb4/7 heterodimer in the context of the complete RNApII complex (2, 3). Rpb4 makes very little contact with the core subunits; the dimer is held primarily through contacts between Rpb7 and core subunits Rpb1 and Rpb6. Rpb4/7 is found near both the transcript-exit groove and the linker to the C-terminal domain (CTD) of Rpb1, a location that would allow for interactions with the nascent RNA transcript as well as protein factors involved in transcription regulation. In fact, Rpb7 contains a potential oligonucleotide-binding domain that faces the presumed RNA exit site (6) and has recently been shown to cross-link to the emerging RNA transcript (31). Recent reports also indicate that the heterodimer can interact with several transcription factors. Rpb7 from the fission yeast Schizosaccharomyces pombe physically interacts in vitro with Seb1, a homolog of the S. cerevisiae CTD-binding protein and termination factor Nrd1 (23). Rpb4 has also been shown to physically interact with Fcp1, a CTD phosphatase (10, 17). Both the capability of Rpb4/7 to interact with these factors and the proximity of the heterodimer to the CTD suggest that Rpb4/7 might play a role in the recruitment of some CTD-binding proteins to transcribing RNApII. Because RPB4 is nonessential in S. cerevisiae, it is possible to examine the role of the Rpb4/7 heterodimer in vivo by using an rpb4Δ strain. Cells that lack RPB4 are both heat and cold sensitive and also grow more slowly than wild-type strains at permissive temperatures (∼24 to 30°C) (32). The sensitivity of rpb4Δ cells to high temperatures has been associated with a general RNApII transcription defect (20, 24). In an attempt to better understand the in vivo effects of Rpb4 loss on the transcribing polymerase, we used chromatin immunoprecipitation (ChIP) to map the cross-linking patterns of polymerase and multiple associated factors along transcribed genes in both wild-type and rpb4Δ strains. Deletion of RPB4 results in decreased polymerase occupancy at the 3′ end of mRNA genes. Additionally, Rpb4 is required for the association of the 3′-end processing factors Rna14 and Rna15 with RNApII and 3′ ends of genes. Our results therefore indicate an in vivo role for Rpb4 in the coupling of transcription and mRNA 3′-end processing.