The CFII components PCF11 and Cbc change subnuclear localization as cells differentiate in an adult stem cell lineage

Abstract

Stage specific increased expression of PCF11 and Cbc, the two components of Cleavage Factor complex II, contribute to developmentally regulated 3'UTR shortening due to alternate polyadenylation of nascent RNA molecules in Drosophila spermatocytes. Here, we show that both Cbc and PCF11 change subnuclear localization during male germ cell differentiation, from homogeneous in the nucleus in spermatogonia and early spermatocytes to concentrated around the nucleolus in later spermatocyte stages.

Figure 1. PCF11 and Cbc change localization from homogeneous in the nucleoplasm to more concentrated at the nucleolar periphery as spermatocytes grow

(A) Schematic of a Drosophila testis. (B) Diagram of the APA switch observed as spermatogonia differentiate into spermatocytes. PASd (orange): the distal PAS processed in spermatogonia. PASp (green): the proximal PAS processed in spermatocytes. The APA switch is at least partially regulated by increased expression of CFII in spermatocytes compared to spermatogonia. (C) Diagram of the cleavage machinery and its complexes: CPSF (yellow), CstF in (blue), CFI (red) and CFII in (purple). UE = Upstream Element, DE = Downstream Element. (D-I) Apical tips of fixed Drosophila melanogaster testes immuno-stained for: (D-D’) PCF11, (E-E’) HA:Cbc, (F-F’) CstF64, (G-G’) CstF50, (H-H’) CPSF6:GFP, (I-I’) PABP2. Scalebars are 100 uM (D-I) and 20 uM (D’-I’). Genotypes: w1118 (D-F-G-I), HA:Cbc (E), CPSF6:GFP (VDRC 318105) (J) Diagram of the heat shock time course system for synchronous spermatocyte differentiation. PHS = post heat shock. (K-O) Apical tips of fixed Drosophila melanogaster testes immuno-stained with antibodies against: PCF11 (green), Fibrillarin (magenta) and VASA (blue). Timepoints: (K) no heat shock, (L) 48H PHS, (M) 72H PHS, (N) 84H PHS, (O) 96H PHS. Scalebars are: 100 uM (K-O) and 20 uM (K’-O’). (P-T) Apical tips of fixed Drosophila melanogaster testes immuno-stained with antibodies against: HA:Cbc (green), and VASA (blue). Timepoints: (P) no heat shock, (Q) 48H PHS, (R) 72H PHS, (S) 84H PHS, (T) 96H PHS. Scalebars are: 100 uM (P-T) and 20 uM (P’-T’).