Interactions of Sen1, Nrd1, and Nab3 with multiple phosphorylated forms of the Rpb1 C-terminal domain in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SEN1 gene codes for a nuclear, ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes, including transcription termination, DNA repair, and RNA processing. Sen1, along with termination factors Nrd1 and Nab3, is required for the termination of noncoding RNA transcripts, but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3, as well as with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II. It has been proposed that Sen1, Nab3, and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser5-phosphorylated (Ser5-P) CTD. To further study the relationship between the termination factors and Rpb1, we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W, a mutation that impairs an interaction between Sen1 and the Ser2-phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser2-P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes, mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser5- to the Ser2-phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes.

Sen1p performs two genetically separable functions in transcription and processing of U5 small nuclear RNA in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SEN1 gene codes for a nuclear-localized superfamily I helicase. SEN1 is an ortholog of human SETX (senataxin), which has been implicated in the neurological disorders ataxia-ocular apraxia type 2 and juvenile amyotrophic lateral sclerosis. Pleiotropic phenotypes conferred by sen1 mutations suggest that Sen1p affects multiple steps in gene expression. Sen1p is embedded in a protein-protein interaction network involving direct binding to multiple partners. To test whether the interactions occur independently or in a dependent sequence, we examined interactions with the RNA polymerase II subunit Rpb1p, which is required for transcription, and Rnt1p, which is required for 3'-end maturation of many noncoding RNAs. Mutations were identified that impair one of the two interactions without impairing the other interaction. The effects of the mutants on the synthesis of U5 small nuclear RNA were analyzed. Two defects were observed, one in transcription termination and one in 3'-end maturation. Impairment of the Sen1p-Rpb1p interaction resulted in a termination defect. Impairment of the Sen1p-Rnt1p interaction resulted in a processing defect. The results suggest that the Sen1p-Rpb1p and Sen1p-Rnt1p interactions occur independently of each other and serve genetically separable purposes in targeting Sen1p to function in two temporally overlapping steps in gene expression.

Detecting phosphorylation-dependent interactions with the C-terminal domain of RNA polymerase II subunit Rpb1p using a yeast two-hybrid assay.

Rpb1p, the largest subunit of S. cerevisiae RNA polymerase II, contains a repetitive structure called the C-terminal domain (CTD). The CTD serves as a scaffold for the regulated association and dissociation of more than a hundred proteins involved in RNA synthesis. Phosphorylation of two serine residues (Ser(2) and Ser(5)) in the repeating units of the CTD change dynamically during the pre-initiation, initiation, elongation and termination of transcription to control the binding and release of transcriptional components. A modification of the well established yeast two-hybrid assay for protein-protein interactions is described that detects interactions between phosphorylated forms of the CTD and proteins whose interactions with the CTD depend on phosphorylation. The efficacy of the approach was established by first showing that two-hybrid fusions containing the CTD are phosphorylated at Ser(2) and Ser(5) residues. Interactions between the CTD and three known CTD-binding proteins were analyzed. The results suggest that the modified two-hybrid system accurately assays CTD-binding and provides a new and convenient assay for CTD-binding proteins.

Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing.

Sen1p in Saccharomyces cerevisiae is a Type I DNA/RNA helicase. Mutations in the helicase domain perturb accumulation of diverse RNA classes, and Sen1p has been implicated in 3' end formation of non-coding RNAs. Using a combination of global and candidate-specific two hybrid screens, eight proteins were identified that interact with Sen1p. Interactions with three of the proteins were analyzed further: Rpo21p(Rpb1p), a subunit of RNA polymerase II, Rad2p, a deoxyribonuclease required in DNA repair, and Rnt1p (RNase III), an endoribonuclease required for RNA maturation. For all three interactions, the two-hybrid results were confirmed by co-immunoprecipitation experiments. Genetic tests designed to assess the biological significance of the interactions indicate that Sen1p plays functionally significant roles in transcription and transcription-coupled DNA repair. To investigate the potential role of Sen1p in RNA processing and to assess the functional significance of the Sen1p/Rnt1p interaction, we examined U5 snRNA biogenesis. We provide evidence that Sen1p functions in concert with Rnt1p and the exosome at a late step in 3' end formation of one of the two mature forms of U5 snRNA but not the other. The protein-protein and protein-RNA interactions reported here suggest that the DNA/RNA helicase activity of Sen1p is utilized for several different purposes in multiple gene expression pathways.