An RNA Polymerase Pause Site Is Associated with the Immunoglobulin μs Poly(A) Site

Abstract

Immunoglobulin μ alternative RNA processing is regulated during B-cell maturation and requires balanced efficiencies of the competing splice (μm) and cleavage-polyadenylation (μs) reactions. When we deleted sequences 50 to 200 nucleotides beyond the μs poly(A) site, the μs/μm mRNA ratio decreased three- to eightfold in B, plasma, and nonlymphoid cells. The activity could not be localized to a smaller fragment but did function in heterologous contexts. Our data suggest that this region contains an RNA polymerase II pause site that enhances the use of the μs poly(A) site. First, known pause sites replaced the activity of the deleted fragment. Second, the μ fragment, when placed between tandem poly(A) sites, enhanced the use of the upstream poly(A) site. Finally, nuclear run-ons detected an increase in RNA polymerase loading just downstream from the μs poly(A) site, even when the poly(A) site was inactivated. When this μ fragment and another pause site were inserted 1 kb downstream from the μs poly(A) site, they no longer affected the mRNA expression ratio, suggesting that pause sites affect poly(A) site use over a limited distance. Fragments from the immunoglobulin A gene were also found to have RNA polymerase pause site activity.

We thank Nick Proudfoot for the α globin and MAZ4 pause sites, the PAC plasmids for pause site analysis, and helpful discussions. We also thank Jeff Wilusz for the ΔU mutation, Chris Milcarek for the γ2b secretory poly(A) site, Cam Dingle and Clarissa Cowan for making and analyzing the stable cell lines, and Cam Dingle, Brett Spear, and Caroline Kane for helpful discussions and comments on the manuscript.

This work was supported by grants MCB-9507513 and MCB-9808637 from the National Science Foundation.