The role of alternative polyadenylation in monocyte:macrophage differentiation Srimoyee Mukherjee and Claire L. Moore Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA Alternative polyadenylation (APA) results in mRNA isoforms that vary in the amount of coding sequence or 3’ UTR regulatory elements. APA is important for response to external signals and differentiation in several cell types, but its role in the differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by both shortening and lengthening of mRNAs in relevant cellular pathways. The proteins of the cleavage/polyadenylation (C/P) machinery increase during differentiation, suggesting a possible mechanism for APA changes. This result was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known APA regulator, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs important for macrophage differentiation and function. Conversely, CstF64 overexpression caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.