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

BackgroundHipfner, Weigmann and Cohen, Genetics, 2002The 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)

bantamhypomorph

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’UTRof 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 sensorin 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 = greenBrdU 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 = greeni.e. bantam expression correlateswith 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?

DAPI-labeled nuclei Activated caspase 3

+ bantam

Expansion of proliferation; reduction in PCD

Fig.6 bantam regulates the hid 3’UTR

hid 3’ UTR

Tubulin-EGFP-hid3’UTR = greenWg protein = red

bantam expression fromEP(3)3622

5 bantam target sitesin melanogaster andpseudoobscura

Fig. 7 bantam Regulates Hid Expression

ptc>hid

ptc>hid+ bantam

Hid antibody AS-RNA probe for hid mRNA

Activated caspase 3antibody

…at the level of protein …and blocks the apoptosis-inducing effects of hid

Fig. 8 bantam levels regulate hid activity in eye development

++ bantam ban-/+