By Mehdi Banan, Ph.D.
Science Content/Business Development Analyst

MicroRNAs (miRNAs) refer to a class of non-protein coding 21-23 bp RNA molecules that are processed from larger (70-90 nt) stem-looped precursors. The first member of this group, lin-4, was identified in C. elegans more than a decade ago. Since then, over 150 miRNAs have been cloned and sequenced in such diverse organisms as worms, fruit flies, humans, and plants (See MicroRNAs are Found in Plants) (1).

While the role of most miRNAs is not known, the functions of a handful of miRNAs have been identified. Some of these miRNAs regulate the expression of target genes by one of two distinct mechanisms. In the first, miRNAs bind to the 3'-untranslated region (3' UTR) of target mRNAs through scattered regions of complementarity (at the beginning and end of this region) to prevent protein synthesis via an unknown mechanism (e.g. lin-4 and let-7 miRNAs in worms) (3). The other mechanism involves miRNA binding to perfectly complementary mRNA sequences to destroy target transcripts (e.g. Scarecrow miRNA in plants). These miRNAs, therefore, function like siRNAs and could be classified as such (2).

Most miRNAs, as in the case of lin-4 and let-7, however, are not perfectly complementary to their target mRNA sequences. Therefore, it has been difficult to identify miRNA targets via alignment programs, which are more adept at identifying long stretches of complementarity. The difficulty in identifying miRNA targets has also made it hard to address miRNA function. Now in the 4 April 2003 issue of Cell, Cohen and colleagues identify the function of a Drosophila miRNA called bantam by taking the opposite approach. Namely, they show that bantam, previously identified as a locus involved in cell proliferation, codes for an miRNA. They then go on to demonstrate that the bantam miRNA inhibits translation of a pro-apoptotic gene called hid. These findings are significant because they help identify not only a novel miRNA, but also its function and one of its target genes (4).

The Approach

Bantam was first identified as a Drosophila locus involved in cell proliferation (5). Previously, it had been determined that several enhancer-containing transposon insertions into a 12.3 kb region spanning this locus caused wing and eye tissue over-growth in fruit flies, presumably due to activation of a nearby gene. In addition, transposon-mediated deletion of a 21 kb fragment spanning this region caused the fruit flies to be smaller than wild-type flies. In order to identify the bantam gene, Cohen and colleagues set out to do complementation tests. The authors were able to complement the 21 kb bantam deletion with a 3.85 kb fragment resulting in "full-sized" fruit flies. Surprisingly, expression of the only identified EST in this region did not complement the bantam deletion.

The authors then set out to identify the gene using a second approach. Specifically, they compared the sequence of this 3.85 kb Drosophila region to that of the malaria mosquito, Anopheles gambiae. As a result, a 90 bp genomic sub-region was identified that was highly conserved between the two organisms. By using an RNA folding program (mfold), it was predicted that this 90 bp region would fold into a stem-looped structure upon transcription. These results suggested that the bantam gene coded for an miRNA. This was confirmed by detection of a ~21 bp bantam miRNA in fruit fly larvae by Northern blot analyses. The role of bantam miRNA in cell proliferation (i.e. tissue over- or under growth) was subsequently confirmed using the 90 bp miRNA precursor in a number of rescue and gain-of-function assays.

The authors then sought to identify bantam mRNA targets. To this end, they used a computational method ("to be described elsewhere") to identify three bantam binding sites in the 3' UTR of hid mRNA (note: hid is a fruit fly gene that induces apoptosis). As expected, they were able to confirm that the bantam miRNA reduces hid translation, but not transcription.

Is bantam an oncogene?

The above results suggest that over-expression of the bantam miRNA promotes cell proliferation by inhibiting translation of the pro-apoptotic gene hid. Deletion of bantam, on the other hand, reduces cell proliferation by allowing hid expression. Together, these results suggest that bantam is a bona fide oncogene. It was, therefore, of great interest to identify bantam homologues in other organisms. The authors conducted a BLAST search of the bantam sequence against other cloned miRNAs. While no matches to human or murine miRNAs were detected, three bantam homologues of unknown function were identified in worms. These results, however, do not preclude the existence of human and mouse miRNAs that are functionally similar to bantam.

The findings by Cohen and colleagues show for the first time that an miRNA can act as an oncogene. Moreover, they suggest that miRNAs might have very diverse biological roles. As stated in a review by Victor Ambros, "one suspects that the diversity and abundance of miRNA genes reflects a broad spectrum of functions and mechanisms, requiring that we approach the study of them with a mindset open to surprise and delight" (6).

References

1. Pasquinelli AE. (2002). MicroRNAs: deviants no longer. Trends in Genet. 18(4): 171-173.
2. Llave C, Kristin ZX, Kasschau KD, Carrington JC. (2002). Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:2053-2056.
3. Lai EC. (2002). Micro RNAs are complementary to 3´ UTR sequence motifs that mediate negative post-transcriptional regulation. Nature Genet. 30:363-364.
4. Brennecke J, Hipfner DR, Stark A, Russell RB, and Cohen SM. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113:25-36.
5. Hipfner DR, Weigmann K, and Cohen SM. (2002). The bantam gene regulates Drosophila growth. Genetics 161:1527-1537.
6. Ambros V. (2001). MicroRNAs: tiny regulators with great potential. Cell 107:823-826.

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