Recent discoveries of small regulatory RNA molecules have been keeping the research community abuzz. Thus far, two classes of tiny regulatory RNA molecules have been identified.

siRNAs
The first class encompasses the small interfering RNAs (siRNAs), which have an integral role in the phenomenon of RNA interference (RNAi), a form of post-transcriptional gene silencing. In RNAi, dsRNAs introduced into certain organisms or cells are degraded into ~22 nt fragments. These ~22 nt siRNA molecules then bind to the complementary portion of their target mRNA and tag it for degradation. siRNAs are believed to have a role in conferring viral resistance and in preventing transposon hopping.

stRNAs
The second class of regulatory small RNAs have been referred to as small temporal RNAs (stRNAs). Examples of RNAs in this group include ~22 nt lin-4 and let-7 RNAs. These RNA molecules, which have a role in the temporal regulation of Caenorhabditis elegans development, are initially processed from a ~70 nt ssRNA transcript folded into a stem-loop structure. Upon processing, these stRNAs are thought to prevent translation of their target mRNAs by binding to the target's complementary 3' untranslated regions (UTRs). Interestingly, the same RNase enzyme, Dicer, processes both siRNAs and stRNAs.

A New Class of Small RNA Molecules
Now a new chapter is added to this story. In 3 papers published in the 26 October 2001 issue of Science (ref. 1-3), nearly 100 additional small ~22 nt RNA molecules have been identified. These RNA molecules, termed microRNAs (miRNAs), were discovered in Drosophila, C. elegans, and HeLa cells by the Bartel, Tuschl and Ambros labs. Much like lin-4 and let-7, these miRNAs are formed from precursor RNA molecules that fold into a stem-loop secondary structure. The newly discovered ~22 nt miRNAs are believed to play a role in regulation of gene expression. Based on the biochemical analysis of stRNAs and siRNAs, it has been hypothesized that miRNAs might regulate translation (like lin-4 and let-7) or modify mRNA stability (like siRNAs).

Identifying miRNAs
The 3 research teams used both a biochemical and a bioinformatics approach to identify these miRNAs. Both siRNAs and stRNAs, which are processed by Dicer, are ~22 nt, have a 5' phosphate and have a 3' hydroxyl group. The Bartel and Tuschl laboratories used a modified directional cloning strategy to select for molecules with similar characteristics. In short, RNA molecules were fractionated by size, ligated to 3' and 5' adapters, reverse transcribed and then amplified by PCR. The resulting PCR products were subcloned and sequenced. The location and clustering of the miRNA precursors within the genome was then determined by querying genomic sequence databases. This analysis also helped determine whether the miRNAs were degradation products of mRNAs, tRNAs, or rRNAs (4).

The Ambrose lab also took a different approach to searching for miRNAs. This lab used the RNA folding program 'mfold' to determine whether highly conserved C. elegans and C. briggsae intergenic sites contained potential miRNA precursors. Northern blots were then used to confirm whether these miRNAs were actually expressed.

miRNA Characteristics
The 3 research teams were able to identify almost 100 new miRNAs. These were comprised of 14 Drosophila, 19 HeLa cell, and 60 C. elegans miRNAs. Approximately 15% of these miRNAs were conserved (with 1-2 mismatches) across worm, fly, and mammalian genomes (4). According to Lau and colleagues in the Bartel lab, however, all miRNAs seemed to have orthologs in other species. All of the identified miRNAs were located at either the 3'- or the 5'-side of a stem loop within a ~70 nt RNA precursor. Some of these precursors were so tightly clustered that it was suggested they might be synthesized on the same transcript.

The expression pattern of the miRNAs varied. While some C. elegans and Drosophila miRNAs were expressed in all cells and at all developmental stages, others had a more restricted spatial and temporal expression pattern. This suggested that these miRNAs, like lin-4 and let-7, might be involved in post-transcriptional regulation of 'developmental' genes.

Stumbling Blocks to Identification and Elucidating Function
Although nearly 100 miRNAs were identified using biochemical and bioinformatics approaches, none of the teams were able to identify the miRNA targets using an informatics approach. The failure to do so may be because the entire miRNA does not have to be complementary to the target in order for binding to occur. The lin-4 and let-7 miRNAs, for example, bind to their respective target mRNAs through complementary 5' and 3' regions (with the middle part of the miRNA looping out). These findings (or lack there-of) leave open the question: what do these miRNAs do? Are they involved in post-transcriptional gene regulation, and if so, what are their target genes?

The isolated miRNAs may constitute only a subset of those available. According to Lau and colleagues, "many of the identified miRNAs were represented by only a single clone". Therefore they hypothesized that their "sequencing had not reached saturation" and that some miRNAs were not isolated in this procedure.

The Tuschl, Bartel and Ambros labs have begun what promises to be a long line of research into miRNAs. It appears that the "tiny RNA world" may not be so tiny after all.

References

1. Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853-858.
2. Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858-862.
3. Lee, R.C. and Ambrose, V. (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862-864.
4. Ruvkun, G. (2001) Glimpses of a tiny RNA world. Science 294:797-799.