We use the human snRNA genes to study fundamental mechanisms of transcription. They represent an excellent model system for this purpose for a number of reasons. First, snRNA genes contain two types of promoters, one type recognized by RNA polymerase (pol) II and one type recognized by pol III. The pol II and III promoters are very similar in structure and indeed they recruit several common transcription factors. Thus, any difference in the initiation complexes assembled on the two types of promoters is likely to be relevant for the determination of RNA polymerase specificity. Second, these promoters are relatively simple. The core pol II snRNA promoters such as the U1 promoter consist of just one essential element, the proximal sequence element or PSE, which recruits a multisubunit factor called SNAPc. The core pol III snRNA promoters such as the U6 promoter contain a PSE as well as a TATA box, which recruits the TATA box binding protein TBP. Both the pol II and III snRNA promoters contain a distal sequence element or DSE, which activates transcription and contains an octamer sequence that recruits the POU domain protein Oct-1 and a so-called SPH site that recruits the zinc finger protein Staf(ZNF143). Third, we have recently identified a set of well-defined factors required and sufficient for transcription from a pol III snRNA promoter. The identification of this basal pol III transcription machinery allows us to study how it is regulated, and indeed we have shown that CK2 can both activate and repress transcription by phosphorylating different targets within this machinery. The identification of this basal pol lll transcription machinery allows us to study how it is regulated. We propose to 1) continue characterizing the mechanisms of U6 transcription including the architecture of SNAPc, the assembly of the U6 transcription initiation complex, and the impact of Oct-1 on the assembly of this initiation complex; 2) characterize how ZNF143 and Oct-1 cooperate to activate U6 transcription from chromatin templates; and 3) determine how and when CK2 regulates pol III transcription from the human U6 promoter. Together, these experiments will reveal how the U6 transcription complex assembles and specifically recruits pol Ill, and how the assembly and activity of the complex is regulated by activators such as Oct-1 and ZNF143, and by kinases such as CK2, during cell growth and proliferation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM038810-18
Application #
6822173
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Tompkins, Laurie
Project Start
1987-07-01
Project End
2008-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
18
Fiscal Year
2004
Total Cost
$245,413
Indirect Cost
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724
Shanmugam, Mayilvahanan; Hernandez, Nouria (2008) Mitotic functions for SNAP45, a subunit of the small nuclear RNA-activating protein complex SNAPc. J Biol Chem 283:14845-56
Yuan, Chih-Chi; Zhao, Xinyang; Florens, Laurence et al. (2007) CHD8 associates with human Staf and contributes to efficient U6 RNA polymerase III transcription. Mol Cell Biol 27:8729-38
Emran, Farida; Florens, Laurence; Ma, Beicong et al. (2006) A role for Yin Yang-1 (YY1) in the assembly of snRNA transcription complexes. Gene 377:96-108
Saxena, Ashish; Ma, Beicong; Schramm, Laura et al. (2005) Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription. Mol Cell Biol 25:9406-18
Hu, Ping; Samudre, Kalpana; Wu, Si et al. (2004) CK2 phosphorylation of Bdp1 executes cell cycle-specific RNA polymerase III transcription repression. Mol Cell 16:81-92
Hu, Ping; Wu, Si; Hernandez, Nouria (2004) A role for beta-actin in RNA polymerase III transcription. Genes Dev 18:3010-5
Hu, Ping; Wu, Si; Hernandez, Nouria (2003) A minimal RNA polymerase III transcription system from human cells reveals positive and negative regulatory roles for CK2. Mol Cell 12:699-709
Ma, Beicong; Hernandez, Nouria (2002) Redundant cooperative interactions for assembly of a human U6 transcription initiation complex. Mol Cell Biol 22:8067-78
Schramm, Laura; Hernandez, Nouria (2002) Recruitment of RNA polymerase III to its target promoters. Genes Dev 16:2593-620
Hu, Ping; Wu, Si; Sun, Yuling et al. (2002) Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits. Mol Cell Biol 22:8044-55

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