ranscription, the initial step in decoding genetic information, has been the intense focus of molecular biologists for several decades. In recent years it has become apparent that many human diseases, including most cancers, many blood and immune disorders, Alzheimer’s disease, Fragile-X syndrome and many others, are a consequence of transcription gone awry. Despite the disparate clinical disorders manifest by these diseases, they share a common feature of aberrant transcription of key genes involved in normal cell growth and function. Accordingly, therapeutic manipulation based on regulation of transcription has become a realistic strategy for treatment of many human disorders.
Transcription and its control have intrigued scientists for another fundamental reason, namely, how it is that a single cell gives rise to a variety of cell types, which all contain exactly the same genetic information. Clearly, organisms are not defined simply by the sequence of their genes, but also by the patterns of gene expression in different cells. This issue is perhaps most evident when considering embryonic stem cells: a study published earlier this year demonstrated that fully differentiated cells from adult human fibroblasts can be reprogrammed to become embryonic stem cells by altered expression of just four transcription factors. This exciting realization has led to new urgency in research aimed at understanding, and ultimately manipulating, the biochemical machinery that controls gene transcription.
My current research efforts are focused on two important questions: 1) how is the processing of messenger RNA coupled to transcription? and, 2) what are the factors involved in this process? The experimental organism used in most of my research is the baker’s yeast Saccharomyces cerevisiae, which enables me to use a powerful combination of classical genetics, molecular biology and modern biochemistry.
The RNAP II transcription cycle
Transcription occurs in distinct stages that include assembly of a preinitiation, promoter melting, initiation, promoter clearance, elongation, termination and reinitiation. Nascent mRNA undergoes modifications that include 5’ capping, splicing, 3’ endonucleolytic cleavage and polyadenylation. Remarkably, these events all occur co-transcriptionally. Progression of RNAPII through the transcription cycle is accompanied by changes in the phosphorylation status of the CTD, a reiterated heptapeptide sequence (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) present at the C-terminus of Rpb1, the largest RNAPII subunit. The CTD appears to function as a platform for the recruitment and exchange of RNA processing factors in a manner dependent upon its phosphorylation status. RNAP II is recruited to the promoter in an unphosphorylated form (RNAPIIA) that becomes extensively phosphorylated (RNAPIIO) during transcription. Ser2 and Ser5 of the CTD are phosphorylated by cyclin-dependent protein kinases: Ser5 is phosphorylated (Ser5-P) by the Kin28 (mammalian CDK7) subunit of TFIIH prior to promoter clearance, whereas Ser2 is phosphorylated (Ser-P) by Ctk1 (mammalian CDK9) during elongation. Ser5-P levels diminish as RNAPII moves into elongation, coincident with Ser2 phosphorylation. Recycling of RNAPII following termination requires dephosphorylation to the IIA form. Several years ago, Fcp1 was discovered as a phosphatase specific for Ser2-P of the CTD. However, the Ser5-P phosphatase, which had to exist, remained elusive.
The Ssu72 RNAPII CTD phosphatase
Using yeast genetic methods, we identified a novel gene, SSU72, based on genetic interaction with TFIIB, an essential transcription initiation factor. The Ssu72 protein was later identified as a component of the 3’-end processing machinery and was found to be required for mRNA 3’-end formation. Furthermore, mutations in SSU72 caused cell growth defects in combination with mutations in genes encoding subunits of RNAP II, and with the genes encoding the Kin28 and Ctk1 CTD kinases. The Ssu72 amino acid sequence includes a CX5RS signature motif of a specific class of protein phosphatases; however, no physiological Ssu72 substrate had been identified. In collaboration with Claire Moore’s research group at Tufts Medical School, I found that Ssu72 is the Ser5-P specific CTD phosphatase and is essential for RNAP II progression through the transcription cycle. The role of Ssu72 in both 3’-end processing and CTD dephosphorylation led us to conclude that it could be one of the factors that couples RNAPII transcription to mRNA processing by recycling RNAPII subsequent to termination. This discovery was cited as one of the most important discoveries in the field of transcription by the “Faculty of 1000.” I also identified the Ssu72 binding motif on CTD, in collaboration with Stewart Shuman’s laboratory at Memorial Sloan Kettering Cancer Center in New York. Recently, Jesper Svestrup’s laboratory in England showed that dephosphorylation of RNAP II Ser5-P by Ssu72 is a prerequisite for RNAP II turnover during transcription of UV-damaged DNA templates, suggesting a role for Ssu72 in skin cancer.
Transcription and proline isomerization
Another interesting structural feature of the CTD is the presence of proline residues immediately adjacent to each of the phosphorylated serine residues. In fact, Pro6 is an essential component of the Ssu72 CTD binding motif. This observation is intriguing because proline residues can adopt either the cis or trans conformation, with dramatically different effects on protein secondary structure. Might the phosphorylation status of the CTD, and ultimately its structure as a platform for the exchange of transcription and processing factors, be affected by proline isomerization?
With this in mind, I asked if Ssu72 functionally interacts with Ess1, a prolyl isomerase essential for yeast cell viability. We were astonished to find (i) that the growth defects of ess1 mutants can be suppressed by overexpression of either Ssu72 or the transcription initiation factor TFIIB; (ii) that ess1 mutants result in accumulation of the Ser5-P form of RNAP II in yeast cells; and (iii) that ess1 mutants are defective for transcriptional activation as well as termination. These results define novel roles for Ess1 in transcription and suggest that it does so by affecting the conformation of the RNAP II CTD.
Ess1 is highly conserved among eukaryotic organisms. Its human ortholog, Pin1, has been implicated in different diseases including cancer and Alzheimer’s disease. Our results support the premise that Ssu72-mediated CTD dephosphorylation is modulated by Ess1-catalyzed CTD prolyl isomerization. We surmise that its action is akin to a “switch” that changes the conformation of the structurally flexible CTD via proline isomerization. In essence, Ess1 renders Ser5-P amenable to the Ssu72 phosphatase, depending on the stage of the transcription cycle. This in turn alters the profile of transcription and RNA processing factors that are recruited to RNAP II. Furthermore, functional interaction of Ess1 with TFIIB links Ess1 to initiation, perhaps by facilitating Ssu72-catalyzed dephosphorylation, converting RNAP IIO to the initiation competent RNAPIIA form.
In summary, progression of RNA polymerase II through the transcription cycle is accompanied by changes in the phosphorylation status of the CTD, a reiterated heptapeptide sequence present at the C-terminus of Rpb1, the largest RNAP II subunit. RNAP II is recruited to the promoter in an unphosphorylated form (RNAP IIA) that is extensively phosphorylated (RNAP IIO) during transcription. Recycling of RNAP IIO following termination requires dephosphorylation to the IIA form. The CTD appears to function as a “control panel” for the recruitment and exchange of transcription and RNA processing factors. Using a combination of classical yeast genetics and modern molecular biology, my research has identified a novel CTD phosphatase, Ssu72, as well as a prolyl isomerase, Ess1, that work together to affect transcription by altering the structure of the CTD. Moreover, Ssu72 and Ess1 are conserved proteins with counterparts in human cells that have been implicated in many different cancers, as well as Alzheimer’s disease.
Krishnamurthy Shankarling has been an adjunct assistant professor in the Department of Biochemistry at UMDNJ-Robert Wood Johnson Medical School since 2005. He received his PhD in biochemistry from Jawaharlal Nehru University, New Delhi, under Professor Rajendra Prasad. His postdoctoral training included a one-year stint at Johns Hopkins University School of Medicine as a postdoctoral fellow, followed by a two-year Alexander von Humboldt fellowship at the University of Duesseldorf, Germany, before joining his current department as a research associate in 2002.