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. 2019 Jul 16;39(15):e00581-18. doi: 10.1128/MCB.00581-18

FIG 2.

FIG 2

In vivo deletion of the polo USE impairs Polo activity and polo pA signal selection. (A) Representative immunofluorescence images and respective quantifications of GFP-Polo, Aurora BT232Ph, and Mps1T490Ph levels at kinetochores of dividing third-instar larval neuroblasts expressing gfp-polo; polo9−/− and gfp-poloΔUSE; polo9−/−. GFP-Polo, Aurora BT232Ph, and Mps1T490Ph fluorescence intensities were determined relative to the Spc105 signal, which was used as a kinetochore reference. All values were normalized to the control (gfp-polo; polo9−/−) mean fluorescence intensity, which was set to 100% (n ≥ 3 kinetochores from at least 10 neuroblasts for each condition). Results are expressed as mean values ± SD, and statistical significance was assessed by the Mann-Whitney test. (B) Representative immunofluorescence images of mitotic neuroblasts from squashed gfp-polo; polo9−/− and gfp-poloΔUSE; polo9−/− larval brains. Spc105 was used as a kinetochore reference. The chromosome content is shown for each representative neuroblast. The graph represents the quantification of chromosome numbers in gfp-polo; polo9−/− and gfp-poloΔUSE; polo9−/− mitotic neuroblasts. (C) Northern blot using total RNA from w1118 (lane 1), gfp-poloΔUSE; polo9/TM6B (lane 2), heph2/TM6B (lane 3), and gfp-poloΔpA1; polo9/TM6B (lane 4) third-instar larva brains. The two endogenous polo mRNAs present in every lane (endogenous polo pA1 and pA2 isoforms) are indicated by arrows. Only the gfp-polo pA2 isoform is expressed by the gfp-poloΔUSE; polo9/TM6B and gfp-poloΔpA1; polo9/TM6B strains, as indicated by the arrowheads. rRNA served as loading control. (D) 3′-end mapping of polo in the abdomens of gfp-poloΔUSE; polo9−/− and gfp-poloΔpA1; polo9−/− adults by 3′RACE. gfp-poloΔUSE; polo9−/− and gfp-poloΔpA1; polo9−/− flies produce two additional polo mRNA isoforms (depicted by * and #) other than the expected short (630-bp) and long (890-bp) mRNAs seen in the control (gfp-polo; polo9/TSTL). NTC, no-template control; and RT−, negative control. In the 3′UTR sequence of polo, the positions of the USE, both pA signals (pA1 and pA2), the cryptic pA1 (AAUAUA) 57 bp upstream of pA1, and the cryptic pA2 (AAUAAU) 113 bp upstream of pA2 are highlighted.