The long term goal of this research effort is to understand the mechanisms by which mRNAs are synthesized in mammalian cells, and to learn how this synthesis is regulated. In particular, we will be concentrating on genes from two cell-studied DNA tumor viruses, SV40 and adenovirus. In this research effort, we will concentrate on several on aspects mRNA synthesis. First, we will exploit our recent observation that exogenously added mRNA precursors can be accurately and efficiently polyadenylated in a HeLa whole-cell lysate. We will study in detail both the genetics and biochemistry of this important reaction. Second, we showed recently that two cloned viral promoters (Ad2 late and SV40 early) have drastically different nucleotide sequence requirements in order to be expressed in two different human cell lines, HeLa and 293. We will determine the genetic basis for these effects, as well as study the biochemical mechanism responsible. Third, we will analyze in depth a series of deletion and point mutations that we constructed in the Ad2 late promoter in order to better understand the molecular basis of transcription initiation and regulation. Fourth, we will study and extend a series of insertion initiation and regulation. Fourth, we will study and extend a series of insertion mutants that we constructed in which the Ad2 late promoter has been inserted at various sites in SV40. These insertions are providing us with important insights into SV40 sites in SV40. These insertions are providing us with important insights into SV40 gene expression and transcriptional control in general. Finally, using antibodies we prepared against HeLa Topoisomerase I, we will study the effects of DNA topology on transcription initiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028983-05
Application #
3276391
Study Section
Molecular Biology Study Section (MBY)
Project Start
1981-04-01
Project End
1989-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
5
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Columbia University (N.Y.)
Department
Type
Graduate Schools
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10027
Ogami, Koichi; Richard, Patricia; Chen, Yaqiong et al. (2017) An Mtr4/ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression. Genes Dev 31:1257-1271
Tian, Bin; Manley, James L (2017) Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol 18:18-30
Morales, Julio C; Richard, Patricia; Patidar, Praveen L et al. (2016) XRN2 Links Transcription Termination to DNA Damage and Replication Stress. PLoS Genet 12:e1006107
Drisaldi, Bettina; Colnaghi, Luca; Fioriti, Luana et al. (2015) SUMOylation Is an Inhibitory Constraint that Regulates the Prion-like Aggregation and Activity of CPEB3. Cell Rep 11:1694-702
Shi, Yongsheng; Manley, James L (2015) The end of the message: multiple protein-RNA interactions define the mRNA polyadenylation site. Genes Dev 29:889-97
Nagaike, Takashi; Manley, James L (2014) In vitro analysis of transcriptional activators and polyadenylation. Methods Mol Biol 1125:65-74
Xiang, Kehui; Tong, Liang; Manley, James L (2014) Delineating the structural blueprint of the pre-mRNA 3'-end processing machinery. Mol Cell Biol 34:1894-910
Di Giammartino, Dafne Campigli; Li, Wencheng; Ogami, Koichi et al. (2014) RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs. Genes Dev 28:2248-60
Morales, Julio C; Richard, Patricia; Rommel, Amy et al. (2014) Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair. Nucleic Acids Res 42:4996-5006
Di Giammartino, Dafne Campigli; Manley, James L (2014) New links between mRNA polyadenylation and diverse nuclear pathways. Mol Cells 37:644-9

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