Coordination of RNA Polymerase II Pausing and 3′ End Processing Factor Recruitment with Alternative Polyadenylation

Abstract

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.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00898-15.

ACKNOWLEDGMENTS

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.

We thank K. Diener, B. Gao, and the UC Denver Nextgen sequencing facility for sequencing, P. Vanlaarhoven for antibody purification, and T. Saldi, R. Sheridan, and M. Cortazar for helpful discussions and comments on the manuscript.