Abstract
Eukaryotic poly(A) signals direct mRNA 3′-end processing and also pausing and termination of transcription. We show that pausing and termination require the processing factor CPSF, which binds the AAUAAA hexamer of the mammalian poly(A) signal. Pausing does not require the RNA polymerase II C-terminal domain (CTD) or the cleavage stimulation factor, CstF, that binds the CTD. Pull-down experiments show that CPSF binds, principally through its 30-kDa subunit, to the body of the polymerase. CPSF can also bind CstF, but this seems to be mutually exclusive with polymerase binding. We suggest that CPSF, while binding the body of the polymerase, scans for hexamers in the extruding RNA. Any encounter with a hexamer triggers pausing. If the hexamer is part of a functional poly(A) signal, CstF is recruited and binds CPSF, causing it to release the polymerase body and move (with CstF) to the CTD.
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Acknowledgements
We thank A. Kazerouninia and F. Rigo for important discussions during the course of this work, S. Vong for comments on the manuscript, C. MacDonald (Texas Tech University) for monoclonal antibodies to CstF64 (3A7) and E1B, R. Krug (University of Texas at Austin) for NS1A expression plasmids and antibody to NS1A, D. Bentley (University of Colorado, Fitzsimons) for B-10 tagged Rpb1 and antibody to CPSF73, C. Milcarek (University of Pittsburg) for antibody to CPSF160, B. Blencowe (University of Toronto) for Flag-tagged Rpb1, and the US National Institutes of Health for grant GM50863.
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A.N. was responsible for all of the experimental work, except for that shown in Fig. 2a and Supplementary Fig. 1a, to which K.N. contributed. A.N. and H.G.M. designed experiments, interpreted data and prepared the manuscript.
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Supplementary information
Supplementary Fig. 1
Effect of NS1A expression on RNA and protein levels. (PDF 1750 kb)
Supplementary Fig. 2
NS1A prevents termination by multiple types of poly(A) signal. (PDF 802 kb)
Supplementary Fig. 3
The CPSF interaction with the polymerase body is robust. (PDF 308 kb)
Supplementary Fig. 4
CPSF30 and CPSF100 bind to the polymerase. (PDF 314 kb)
Supplementary Fig. 5
Specificity of the antibody to CPSF (N-20) used in Figures 5 and 6. (PDF 296 kb)
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Nag, A., Narsinh, K. & Martinson, H. The poly(A)-dependent transcriptional pause is mediated by CPSF acting on the body of the polymerase. Nat Struct Mol Biol 14, 662–669 (2007). https://doi.org/10.1038/nsmb1253
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DOI: https://doi.org/10.1038/nsmb1253
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