(from the application): PolyA-dependent termination is a process that is orchestrated by the polyA signal and that involves multiple transcription and RNA processing components within a large rnRNA """"""""factory."""""""" The signal to terminate is generated during the assembly of the cleavage and polyadenylation apparatus. Using cis-antisense rescue for measuring the rate of 3'-end processing complex assembly in vivo we will ask: 1) Does assembly rate govern polyA site strength? and 2) Does assembly occur by stepwise addition of factors, or via a holocomplex? The ability of the polyA signal in the RNA to direct termination requires that 3'-end processing events be coupled to transcriptional events. How coupling is achieved is not known. We propose a model that is consistent with all available information. The main premise of the model is that coupling is achieved through intimate involvement of the polymerase with the assembly of the cleavage and polyadenylation apparatus. The model predicts unusual interactions between polyA sites located close to each other in vivo. For example, the total cleavage and polyadenylation activity for two strong polyA sites in tandem should decrease if the first site is weakened by mutation, but then increase again if the first site is completely inactivated. On the other hand, the model predicts that polyA sites will have no effect on one another when allowed to undergo processing in the absence of transcription in vitro. Experiments are proposed to test the various predictions of the model. Nothing is known of the molecular events involved in polyA-dependent termination because no system has yet been developed for studying polyA-dependent termination in vitro. We have now developed such a system and propose experiments to answer the following questions: 1) Is the processing factor, CPSF, required for polyA-dependent termination? 2) Is the transcription factor, TFIID, required to recruit CPSF? 3) Is termination mechanistically coupled to extrusion of the polyA signal? 4) How far must the polyA signal be extruded in order to function in driving termination? 5) At what point does the transcription elongation complex acquire termination competence? 6) At what point does the complex acquire the factors uniquely required for termination? Finally, methods for identifying specific factors involved in polyA-dependent termination are proposed. First, various direct approaches are presented for isolating termination factors from crude extracts. Then the possibility is considered of using immunodepletion to identify polyA-dependent termination factors from among proteins that have already been described.

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National Institute of General Medical Sciences (NIGMS)
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Cell Development and Function Integrated Review Group (CDF)
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Tompkins, Laurie
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University of California Los Angeles
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Rigo, Frank; Martinson, Harold G (2009) Polyadenylation releases mRNA from RNA polymerase II in a process that is licensed by splicing. RNA 15:823-36
Rigo, Frank; Martinson, Harold G (2008) Functional coupling of last-intron splicing and 3'-end processing to transcription in vitro: the poly(A) signal couples to splicing before committing to cleavage. Mol Cell Biol 28:849-62
Nag, Anita; Narsinh, Kazim; Martinson, Harold G (2007) The poly(A)-dependent transcriptional pause is mediated by CPSF acting on the body of the polymerase. Nat Struct Mol Biol 14:662-9
Nag, Anita; Narsinh, Kazim; Kazerouninia, Amir et al. (2006) The conserved AAUAAA hexamer of the poly(A) signal can act alone to trigger a stable decrease in RNA polymerase II transcription velocity. RNA 12:1534-44
Rigo, Frank; Kazerouninia, Amir; Nag, Anita et al. (2005) The RNA tether from the poly(A) signal to the polymerase mediates coupling of transcription to cleavage and polyadenylation. Mol Cell 20:733-45
Park, Noh Jin; Tsao, David C; Martinson, Harold G (2004) The two steps of poly(A)-dependent termination, pausing and release, can be uncoupled by truncation of the RNA polymerase II carboxyl-terminal repeat domain. Mol Cell Biol 24:4092-103
Kim, Steven J; Martinson, Harold G (2003) Poly(A)-dependent transcription termination: continued communication of the poly(A) signal with the polymerase is required long after extrusion in vivo. J Biol Chem 278:41691-701
Orozco, Ian J; Kim, Steven J; Martinson, Harold G (2002) The poly(A) signal, without the assistance of any downstream element, directs RNA polymerase II to pause in vivo and then to release stochastically from the template. J Biol Chem 277:42899-911
Tran, D P; Kim, S J; Park, N J et al. (2001) Mechanism of poly(A) signal transduction to RNA polymerase II in vitro. Mol Cell Biol 21:7495-508
Chao, L C; Jamil, A; Kim, S J et al. (1999) Assembly of the cleavage and polyadenylation apparatus requires about 10 seconds in vivo and is faster for strong than for weak poly(A) sites. Mol Cell Biol 19:5588-600

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