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

MicroRNAs (miRNAs) refer to a class of non-coding 20-24 nt RNA molecules that are processed from larger stem-looped precursors. So far, over 150 miRNAs have been identified in fruit flies, worms, and animals (1,2) (Also see MicroRNAs (miRNAs): A New Class of Small RNA Molecules). Now in the 1 July, 2002 issue of Genes & Dev., David Bartel and colleagues show that miRNAs are also present in plants (3). The new findings imply that miRNAs have arisen in the early stages of eukaryotic evolution. Moreover, their existence across different kingdoms suggests that they may play important regulatory roles in both plants and animals.

Isolation of Plant miRNAs

Bartel and colleagues used a differential cloning strategy to isolate 16 miRNAs from the plant, Arabidopsis thaliana. In brief, plant total RNA was run on a gel. RNA species of 18-24 nt were excised from the gel, purified, ligated to 5´ and 3´ adapters, and amplified by RT-PCR. The amplified fragments were then subcloned into plasmids and sequenced. Only sequences represented by more than one clone were considered for further analysis (to avoid inclusion of mRNA degradation products). Using this approach, 16 plant miRNAs were identified. Importantly, it was shown that all 16 miRNAs are in genomic regions that could potentially code for larger stem-looped precursors (the actual precursors were not detected, possibly due to high turnover rates).

The Role of Plant miRNAs

The role of plant miRNAs is not clear. However two pieces of evidence suggested that they might play roles in plant development. First, the authors showed that expression levels of 3 miRNAs are reduced in carpel factory (car) mutant plants. CARPEL FACTORY is a DICER-like enzyme that is involved in plant development. When car is mutated, the plants show defects in embryo and leaf development. The results by Bartel and colleagues suggest that these defects result from a reduction in miRNA-processing.

In addition, the authors showed that 15 of the 16 identified plant miRNAs have higher expression levels in particular plant tissues. These results further suggest that miRNAs play a role in the development of plant tissues.

Targets for Plant miRNAs

It been proposed that miRNAs inhibit translation by binding to complementary region(s) of target mRNAs. This assumption has stemmed from findings that lin-4 and let-7, two miRNAs originally identified in C. elegans, are partially complementary to the 3´ untranslated region (3´ UTR) of their target transcripts (lin-14 and lin-41, respectively). Similar partial homologies have also been found between a number of Drosophila miRNAs and the 3´ UTR of their potential mRNA targets (4).

Lack of complete homology between miRNAs and their targets has made target-identification difficult. The authors also had difficulty in identifying most of the putative plant-miRNA targets. They did, however, find one plant miRNAs that showed perfect complementarity to 3 potential target genes. These corresponded to scarecrow genes, which code for putative transcription factors. At present, it is not clear whether these genes are genuine miRNA targets. Nevertheless, this is the first time that a perfect complementarity between a miRNA sequence and its putative target has been found.

It is interesting to note that siRNAs, which are also 21-25 nt in length, bind to complementary target transcripts and tag them for destruction. Therefore it is possible that the above plant miRNA, which has perfect homology to its putative targets, functions like an siRNA (Also see RNA Interference and Gene Silencing: History and Overview)

What the Future Holds?

It is evident that miRNAs are expressed in a wide variety of organisms and even across kingdoms. Yet the major unanswered question is: what is their function? In order to answer this question, plant and worm genomes could be scanned for mutations at or near miRNAs. Alternatively, miRNA sequences could be deleted by targeted-disruption in fruit flies. (2). Organisms that contain mutated miRNAs could then be studied for accompanying phenotypic defects. Such endeavors, which are already underway, should help decipher the function of miRNAs.

As mentioned by Phillip Zamore, "It would be a cruel joke that nature has played on scientists if the [miRNA] sequences had been conserved in evolution but with no function." (5).

References

1. Schwarz DS and Zamore PD. (2002). Why do miRNAs live in the miRNP? Genes & Dev. 16:1025-1031.
2. Pasquinelli AE. (2002). MicroRNAs: deviants no longer. Trends in Genet. 18(4):171-173.
3. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, and Bartel DP. (2002). MicroRNAs in plants. Genes & Dev. 16:1616-1626.
4. Lai EC. (2002). Micro RNAs are complementary to 3´ UTR sequence motifs that mediate negative post-transcriptional regulation. Nature Genet. 30:363-364.
5. Dennis, C. (2002). The brave new world. Nature 418:122-124.

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