BIOLOGICAL OVERVIEW

Growth of tissues and organs during animal development involves careful coordination of the rates of cell proliferation and cell death. Cell proliferation depends on signals to stimulate cell growth and cell division. In addition, cells compete for intercellular survival signals which are required to prevent them from undergoing apoptosis in response to growth stimuli. How these cellular processes are coordinated with pattern formation during animal development is a challenging question in developmental biology. The bantam gene of Drosophila has been found to encode a 21 nucleotide microRNA (miRNA) that promotes tissue growth. bantam expression is temporally and spatially regulated in response to patterning cues. bantam microRNA simultaneously stimulates cell proliferation and prevents apoptosis. The pro-apoptotic gene hid has been identified as a target for regulation by bantam miRNA, providing an explanation for bantam's anti-apoptotic activity (Brennecke, 2003).

Secreted signaling proteins of the Hedgehog, Wingless/Wnt, and Dpp/BMP families control spatial pattern formation during animal development. Evidence has also begun to accumulate implicating these signaling proteins in control of imaginal disc growth during Drosophila development. Hedgehog and Dpp signaling have been shown to stimulate cell proliferation in the imaginal discs of Drosophila. Dpp is also thought to provide a survival signal. Notch and Wingless signaling are required for tissue growth and cell survival, and in some circumstances direct exit from cell proliferation. At the cellular level, the functions of a number of genes involved in control of cell growth and division have been characterized in the imaginal discs, including components of the Insulin/PI3Kinase pathway, the Myc, Ras and E2F oncogenes, and Cyclin D/CDK4. In spite of this considerable progress, how intercellular signals coordinate pattern formation with cell proliferation and cell survival remains poorly understood (Brennecke, 2003 and references therein) (Brennecke, 2003).

The bantam locus of Drosophila was identified in a gain-of-function screen for genes that affect tissue growth (Hipfner, 2002). Evidence that the bantam gene encodes a 21 nucleotide microRNA (miRNA). miRNAs are small RNAs, typically of 21-23 nucleotides, that direct posttranscriptional regulation of gene expression. miRNAs are excised by the Dicer RNase complex from longer precursor RNAs that form imperfect hairpin structures. Typically only one arm of the hairpin is recovered as a mature product. Processing of miRNAs has much in common with the production of the short interfering RNAs that direct RNA-mediated interference (RNAi) (Brennecke, 2003 and references therein).

Two mechanisms for regulation of gene expression by miRNAs have been reported. Target RNAs containing sequences perfectly complementary to the miRNA are cleaved by ribonucleases in the RISC complex, as described recently for transcripts encoding scarecrow-like family transcription factors in plants (Llave, 2002). Target RNAs containing sequences imperfectly complementary to the miRNA can be subject to translational control. The let-7 and lin4 miRNAs of C. elegans (see Drosophila let-7) act in this manner to repress translation of several mRNAs, which control the transitions between larval stages. Hundreds of miRNAs have been identified in plants and animals. Many of these are conserved between closely related species, and some across phyla. Such numbers suggest that miRNAs have diverse and important regulatory roles in organisms. However, apart from the few exceptions mentioned above, their functions are unknown (Brennecke, 2003 and references therein). The findings of this study assign a role to the miRNA encoded by the bantam gene in control of cell proliferation and apoptosis during Drosophila development. Further, they provide a link between the mechanisms that control patterning and tissue growth during animal development (Brennecke, 2003).

Evidence has been found that the bantam gene of Drosophila encodes a miRNA that controls cell proliferation and apoptosis. bantam-induced tissue growth results from an increase in cell number due to an increase in the rate of cell proliferation, not from an increase in cell size (Hipfner, 2002). The anti-apoptotic effects of bantam are not sufficient to explain its effects on tissue growth. Expression of the caspase inhibitor P35 effectively blocks apoptosis in vivo but does not cause net tissue growth (Brennecke, 2003).

bantam's effects are distinguishable from those of Ras, Myc, and the insulin/PI3K pathway, which primarily affect cellular growth. The effects of bantam most closely resemble those caused by altered levels of cyclinD/CDK4 activity. Like cyclinD/CDK4, bantam controls cellular growth and cell cycle progression in a coordinated manner (Brennecke, 2003).

To understand how bantam miRNA promotes cell proliferation and prevents cell death, it will be necessary to identify the genes that it regulates. Using a computational method for predicting possible target genes of miRNAs, the pro-apoptotic gene hid has been identified as a direct target for regulation by bantam miRNA, suggesting one mechanism by which bantam contributes to controlling cell death. bantam target sequences were not found in any of the following genes: ras, myc, dE2F, dDP, cyclin D, CDK4, cyclin E, string, or in components of the insulin/PI(3) kinase pathway. If it is assumed that bantam acts as a negative regulator of target genes (as for hid), its targets might be negative regulators of cell growth or cell proliferation. No bantam target sites were found in the following genes: PTEN, TSC1, TSC2, salvador, warts/lats, merlin, expanded, lethal giant larvae, discs large, and discs overgrown. It seems likely that bantam may regulate an as yet unidentified negative regulator of cell proliferation. In depth analysis of additional predicted targets will be required to determine how bantam controls cell proliferation (Brennecke, 2003).

The three proapoptotic genes hid, reaper, and grim downregulate levels of the IAP proteins in Drosophila, thereby preventing caspase activation. Unlike reaper and grim, whose activity appears to be regulated primarily at the transcriptional level, hid mRNA is also detected in cells that do not undergo apoptosis. Evidence has been presented for transcriptional regulation of hid and for posttranslational regulation of Hid activity by the MAPK signaling pathway. By showing that bantam blocks the activity of Hid(Ala5), which is insensitive to MAPK regulation, an indirect effect of bantam mediated by regulation of the MAPK pathway is excluded. The hid 3'UTR confers bantam-mediated regulation on a heterologous reporter. These findings provide evidence that hid is subject to translational regulation in vivo by the bantam miRNA (Brennecke, 2003).

hid is known to play an important role in regulating apoptosis in eye development. Removing one copy of the endogenous bantam gene enhances the severity of the Hid-induced apoptosis phenotype in the eye, whereas the severity of the reaper-induced apoptosis phenotype is affected much less strongly. Similarly, overexpression of bantam suppresses both the GMR-hid and GMR-reaper phenotypes, but has a stronger effect on hid. The severity of the GMR-reaper phenotype is sensitive to the levels of hid activity. By overexpressing bantam, Hid levels are reduced, providing an explanation for the observed suppression of the GMR-reaper phenotype. Similarly, by removing one copy of bantam an increase in endogenous hid activity in the eye would be expected. By altering the level of Hid, bantam can indirectly alter the threshold for reaper-induced apoptosis. This provides an explanation for the slight increase in severity of the GMR-reaper phenotype observed. There are no bantam target sites in the reaper gene, suggesting that bantam's effect on the GMR-reaper phenotype must be indirect. Finally, no increase in apoptosis was observed in bantam mutant clones in the wing disc. Endogenous hid has not been implicated in developmental control of cell death in the wing (Brennecke, 2003).

The secreted signaling proteins that control spatial pattern during Drosophila development also control tissue growth. Hedgehog promotes cell growth and proliferation in the eye and wing discs by inducing cyclin D and cyclin E expression. The Dpp morphogen gradient has been implicated in control of proliferation and appears to provide cell survival cues as well. Wingless appears to act as both a positive and negative regulator of growth in a context-dependent manner in the wing disc. Evidence is presented that blocking Wnt signaling by overexpression of a dominant-negative form of the Fz2 receptor protein restores cell proliferation in the ZNC, at least in part by regulating bantam miRNA expression. It will be of interest to learn whether bantam homologs play a comparable role as regulators of cell proliferation during vertebrate embryogenesis. BLAST searches did not find sequences identical to the bantam miRNA in the mouse or human genomes, however the possibility exists that a functionally homologous miRNA may differ slightly in nucleotide sequence. The putative Anopheles homolog has a single nucleotide alteration. In addition, three cloned C. elegans miRNAs, mir80, mir81, and miR82, are similar to bantam. It remains to be determined if they have a similar function (Brennecke, 2003).

Is bantam an oncogene? The possibility that misexpression or misregulation of bantam homologs might be responsible for diseases of cell proliferation is intriguing. bantam can suppress apoptosis while stimulating cell proliferation in Drosophila. In Drosophila, cyclinD/CDK4 overexpression causes a proliferation phenotype similar to bantam; cyclinD/CDK4 human homologs are oncogenes. Oncogenes such as Myc and E2F promote apoptosis as well as cell proliferation. Tumor formation requires additional anti-apoptotic inputs. bantam is able to do both and so its putative vertebrate homologs may be oncogenes. In this context, it is noteworthy that Argonaute family genes, which encode components of the cellular machinery needed to produce miRNAs, have been implicated in numerous biological processes including tumorigenesis (Brennecke, 2003).

miRNAs are short noncoding sequences and so present small targets for chemical mutagenesis. Identification of bantam depended on a gain-of-function genetic strategy making use of EP elements to overexpress genes tagged by P element insertion. Several other miRNAs are located next to EP elements in the Drosophila genome, opening the possibility for analysis of their functions in vivo (Brennecke, 2003).

GENE STRUCTURE

The bantam region does not have the capacity to encode a protein with significant sequence similarity to proteins in other genomes examined. A BLAST search of the Anopheles gambiae genome with the bantam region identified a sequence with 30/31 identical residues located 302 nucleotides (nt) downstream from RE64518. Alignment of the two genomic regions containing these sequences with ClustalW identified a block of ~90 residues with considerable similarity. The Drosophila and Anopheles sequences were each predicted to fold into stable hairpin structures using mfold (www.bioinfo.rpi.edu/applications/mfold/old/rna/). The region of highest similarity between these sequences was found on the same arm of the hairpin. These observations suggested that the predicted hairpins might be precursors in the production of a microRNA (Brennecke, 2003).

Indeed, a small RNA of ~21 nt was detected by Northern blot analysis of total RNA from third instar larvae, using an end-labeled probe complementary to the conserved 31 nt sequence. The other arm of the hairpin did not produce a detectable miRNA. bantam miRNA levels were elevated in total RNA from actin-Gal4 > EP(3)3622 larvae and by Gal4-directed expression of the 6.7 kb BamHI genomic rescue fragment. A larger product was also detected, which may represent the hairpin precursor. To define more precisely the sequences necessary to produce the bantam miRNA, a 584 nt fragment containing the predicted hairpin was cloned into the 3'UTR of a heterologous transcript (UAS-C). Expression of UAS-C under engrailed-Gal4 control also led to overproduction of bantam miRNA. bantam miRNA was absent from larvae homozygous for the bantam^Δ1 deletion. Both products were detected in Schneider S2 cells. S1 nuclease mapping was used to identify the 5' and 3' ends of the miRNA. The deduced product is the 21 nt miRNA 5'-GUGAGAUCAUUUUGAAAGCUG (Brennecke, 2003).

See Sanger center's miRBase for bantam Stem-loop sequence.

date revised: 16 August 2003

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