bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila Julius Brennecke, David Hipfner, Alexander Stark, Robert Russell, and Stephen M. Cohen Cell, Vol. 113, 25-36, April 4, 2003. European Molecular Biology Laboratory, Heidelberg, Germany

Background Hipfner, Weigmann and Cohen, Genetics, 2002 The bantam Gene Regulates Drosophila Growth performed a gain-of-function screen using the EP method (Rorth, 1996) screened for GAL4-dependent effects on tissue size without disrupting pattern identified a locus that they called bantam

Background continued identified 3 EP lines in a 12 kb interval at 61C7-8 P-element-mediated excision of one EP was used to create a deletion (21 kb) deletion is homozygous lethal at early puapl stages; mutant larvae lack detectable imaginal discs Deletion/P allele animals are viable but 15% smaller small size is due to a reduction in cell number, not cell size overexpression of bantam causes overgrowth of tissue ( wing and eye) no predicted genes within the surrounding 41 kb interval Expression levels (by Northern) of neighbouring genes appeared normal in the bantam deletion

Figure 1. Map of the bantam Locus 41 kb region (btn predicted genes) bantam hypomorph Overlap region BLAST: Drosophila vs. Anopheles Mfld (www.bioinfo.rpi.edu/….)

Figure 2. Bantam Encodes a miRNA 3rd instar larval RNA (Northern) 21nt 584nt 100nt in 3’UTR of UAS-EGFP S1 nuclease –protection mapping

Figure 3. Bantam miRNA expression Tub—GFP—ban,ban(target) Tub—GFP—UTR Upregulation of bantam sensor in clones of mutant cells Downregulation of bantam sensor in clones overexpressing bantam Mutant clones smaller in size than wild-type twins; bantam acts cell autonomously

Fig.4 bantam Expression and Cell Proliferation bantam sensor = green BrdU incorporation - purple Proliferating cells are correlated with increased bantam miRNA activity (i.e. decreased sensor) Larval brain Wing disc (ZNC = Zone of Non-proliferating Cells) ptc-Gal4 x UAS-C + GFP = green i.e. bantam expression correlates with cell proliferation

Fig.5 bantam inhibits proliferation-induced apoptosis studies on Myc and E2F oncogenes have shown that strong proliferative stimuli induce apoptosis; Cell proliferation results only when apoptosis is simultaneously prevented. Can bantam suppress proliferation-induced apoptosis caused by overexpression of E2F and its cofactor DP? Activated caspase 3 DAPI-labeled nuclei + bantam Expansion of proliferation; reduction in PCD

Fig.6 bantam regulates the hid 3’UTR 5 bantam target sites in melanogaster and pseudoobscura Tubulin-EGFP-hid3’UTR = green Wg protein = red bantam expression from EP(3)3622

Fig. 7 bantam Regulates Hid Expression Hid antibody AS-RNA probe for hid mRNA Activated caspase 3 antibody ptc>hid ptc>hid + bantam …at the level of protein …and blocks the apoptosis-inducing effects of hid

Fig. 8 bantam levels regulate hid activity in eye development