Most mammalian genes produce transcripts whose 3' ends are processed at multiple alternative positions by cleavage/polyadenylation (CPA). Poly(A) site cleavage frequently occurs cotranscriptionally and is facilitated by CPA factor binding to the RNA polymerase II (Pol II) C-terminal domain (CTD) phosphorylated on Ser2 residues of its heptad repeats (YS2PTSPS). The function of cotranscriptional events in the selection of alternative poly(A) sites is poorly understood. We investigated Pol II pausing, CTD Ser2 phosphorylation, and processing factor CstF recruitment at wild-type and mutant IgM transgenes that use alternative poly(A) sites to produce mRNAs encoding the secreted and membrane-bound forms of the immunoglobulin (Ig) heavy chain. The results show that the sites of Pol II pausing and processing factor recruitment change depending on which poly(A) site is utilized. In contrast, the extent of Pol II CTD Ser2 phosphorylation does not closely correlate with poly(A) site selection. We conclude that changes in properties of the transcription elongation complex closely correlate with utilization of different poly(A) sites, suggesting that cotranscriptional events may influence the decision between alternative modes of pre-mRNA 3' end processing.

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Published in Molecular and Cellular Biology, v. 36, no. 2, p. 295-303.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The copyright holders have granted the permission for posting the article here.

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Funding Information

This work was supported by NIH grant GM58613 to D.L.B. and NSF grant MCB-0919099 to M.L.P. S.K. was supported by a postdoctoral fellowship (grant PF-07-297-01-GMC) from the American Cancer Society and the Clark family. B.F. was supported by the Victor W. Bolie and Earleen D. Bolie Graduate Scholarship Fund through the UC Denver Molecular Biology Program.

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Supplemental File 1 - Fig. S1-S4, and Table S1