Our laboratory studies the regulation of gene expression in eukaryotic organisms. We are especially interested in transcription of protein-encoding genes by RNA polymerase II (Pol II). The experimental organism used in most of our work is the yeast Saccharomyces cerevisiae, which enables us to use a powerful combination of classical genetics, molecular biology and modern biochemistry in our research. Our current efforts are focused on two questions: 1) How is the transcription coupled to RNA processing? and 2) How do gene loops that juxtapose the 3'-end of the gene with the promoter affect the mechanism and regulation of gene expression?
1) Coupling of 3' end processing to Pol II transcription. Nascent mRNA undergoes modifications that include 5' capping, splicing, 3' endonucleolytic cleavage and polyadenylation. These processing events occur co-transcriptionally and involve recruitment and exchange of processing enzymes to the C-terminal domain (CTD) of the Rpb1 subunit of Pol II. The CTD is phosphorylated and dephosphorylated at Ser2 and Ser5 during the transcription cycle. We recently discovered that the Ssu72 protein is an integral component of the CPF 3' end processing complex and is a CTD Ser5-P phosphatase. We are now focused on (i) determining how Ssu72-mediated Ser5-P dephosphorylation affects Pol II progression through the transcription cycle; (ii) how Ssu72 is regulated by the transcriptional machinery; and (iii) how Ssu72 is regulated by 3' end processing factors. These questions are being addressed in collaboration with Professor Claire Moore (Tufts Medical School).
2) Role of "gene loops" in transcription. Although Ssu72 is a component of the CPF 3' end processing complex, we first identified this protein based on genetic and physical interactions with TFIIB, a transcription initiation factor. As such, Ssu72 defined an unexpected link between the Pol II initiation and termination machineries. This suggested to us that the ends of gene might physically interact to form gene loops. Our recent studies revealed that gene loops are a general feature of Pol II transcription. Looping is dependent upon transcription and requires specific components of the transcription initiation and 3'-end processing complexes, including the Ssu72 CTD phosphatase. We are now working (i) to define the factors and mechanisms involved in loop formation; and (ii) to determine the functional significance of gene loops with respect to regulation of gene expression.
A remarkable feature of Pol II transcription and mRNA processing is the extent to which these two coupled processes – and the proteins that facilitate them – are conserved among eukaryotic organisms. Accordingly, we are able to exploit the extensive arsenal of experimental approaches available in yeast with the results directly applicable to human biology and medicine.
