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Fig. 7. BrunoL1 function is dependent on BREs in the 3′UTR. (A) Schematic diagram illustrating the sequence of the 3 BREs in the 3′UTR of cyclin A2 and the respective point mutations made. (B) Injection of gfp-bre resulted in a low level of GFP fluorescence. (C) Co-injection of brunol1 increased GFP fluorescence in 78% of embryos (n=67). (D) Injection of the gfp-bremut mRNA produced a similar low-level fluorescence as seen with the gfp-bre mRNA. (E) No increase in GFP fluorescence was seen when brunol1 was co-injected with gfp-bremut mRNA (n=17). (F) Quantification of GFP fluorescence. Injection of brunol1 mRNA along with gfp-bre resulted in a 5-fold increase in GFP fluorescence. For details on how we calculated fold stimulation see materials and methods. Experiment repeated at least 3 times. (G) RT-PCR detection of gfp, edd and cyclin A2 from total RNA of injected embryos (1st four lanes) or from total RNA after immunoprecipitation using flag antibody (2nd four lanes). gfp mRNA was immunoprecipated in the presence of Flag-BrunoL1 protein, but not when the BRE sites were mutated (BREmut+BrunoL1). Edd was used as a control, and was detected in total RNA, but not in the immunoprecipitated RNA. Endogenous cyclin A2 mRNA was detected in both lanes where BrunoL1 was immunoprecipitated. (H) Levels of endogenous Cyclin A2 protein in control and brunol1 injected embryos, demonstrating that there was increased Cyclin A2 protein levels in brunol1 injected embryos. Image published in: Horb LD and Horb ME (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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