Despite the recent progress, most miRNA researchers will agree that we have insufficient knowledge of how miRNAs identify their targets in vivo
. This makes it a tough assignment for the informatician to assemble the desired program to predict targets. Two recent studies provide the first systematic studies of the requirements for miRNA: mRNA-target pairing [26
]. Several unexpected insights emerge that support the notion that miRNA:target interaction is not a simple consequence of nucleic-acid hybridization. Nevertheless, the conclusions are not wholly consistent, indicating that a complete understanding of miRNA target selection is still to come.
The data [26
] provide experimental support for the idea that pairing to the 5' region of the miRNA is a major determinant in target selection (Figure ). But a surprise was that the presence of G:U base-pairs in the 5' miRNA region decreased target regulation far above its thermodynamic effect on duplex formation [28
]. Thus, predicted targets from schemes that allow for G:U or mismatched base-pairs in this region cannot be considered equivalent to those with perfect Watson-Crick pairing. Nevertheless, G:U base-pairs to the miRNA-targeting region cannot be totally discounted either. For example, demanding Watson-Crick pairing to the miRNA-targeting region causes one to miss the perfect complementarity between miR-196
and its bona fide
]. A further complication is that certain functional miRNA-binding sites (including one of the let-7
sites in lin-41
) actually contain a bulge in the middle of the targeting region [10
]. This phenomenon needs to be investigated further to resolve it with the so-called 5'-pairing rule. A possible resolution may be that 5' broken sites (Figure ) are nonfunctional unless extensive 3' pairing is present.
Another controversial point regards the general appearance of a miRNA-binding site (Figure ). One study concluded that miRNA-binding sites consist of an RNA duplex with a central bulge of prescribed lengths on either the miRNA or target side [26
], a description that certainly fits some of the published target sites. But the other study [28
] demonstrated that pairing to the 3' region of the miRNA could be entirely eliminated with minimal effects on target regulation (although strong 3' pairing became important in 5'-weak cases) [28
]. This challenges common assumptions that target recognition involves an RNA duplex along the length of the miRNA and that greater complementarity indicates a better miRNA-binding site. Could it be that as little as eight nucleotides of complementarity to the 5' end of an miRNA suffices for regulation? This might be consistent with the growing appreciation of extensive 'off-target' regulation of modestly complementary transcripts by the small interfering RNAs (siRNAs) involved in RNAi [30
]. These differing views might potentially be reconciled if miRNA-mediated regulation by multiple sites is governed primarily by the 5'-pairing rule whereas regulation by a single site might necessitate more extensive and/or specific pairing configurations (Figure ).
Other issues remain to be resolved. Foremost among these are the factors contributing to site insufficiency. Insertion of miRNA-binding sites into a heterologous context often suffices to bring a transcript under miRNA control, but a significant fraction of tested sites fail for unknown reasons. More importantly, some in vivo
tests suggest that target regulation is not always so forgiving of context. Specifically, mutation of sequences in the lin-41
3' UTR in between two bona fide let-7
sites renders lin-41
nonresponsive to let-7
in transgenic worms [33
]. Moreover, certain multimers of two let-7
or six lin-4
binding sites fail to mediate appropriate regulation in vivo
]. Understanding why these site configurations do not work may improve identification of 'real' sites. For example, these failures might be due to influences of 3' UTR structure on miRNA accessibility, or the necessity for co-regulation by other factors - potentially even other miRNAs. Germane to the latter possibility is understanding the functional interactions between binding sites for the same miRNA and for different miRNAs in an individual target transcript (Figure ), either of which could have synergistic consequences on net regulation [28
]. Another unanswered question is whether animal miRNA-binding sites can reside in coding regions or 5' UTRs, which are usually excluded from analyses. Indeed, the general possibilities that miRNAs might regulate noncoding RNAs or even DNA have been little explored [35
Two additional biological considerations need to be included in the prediction of miRNA targets. Firstly, with the exceptions of lsy-6
, and bantam
], we are generally ignorant of the spatial expression of miRNAs on a cell-by-cell basis. This means that we do not generally know that any miRNA and its predicted target are ever present in the same cell, an obvious prerequisite for a regulatory relationship. Secondly, we generally lack information on the relative levels of miRNA and target on a per-cell basis. The study by Doench and Sharp [28
] showed that target regulation that can be detected when the miRNA is very abundant does not occur when the miRNA is rarer [28
]. Thus, greater biological relevance to target prediction may come from incorporating data on miRNA:target coexpression and relative levels. Finally, improvements will come from having additional genomes sequenced, which will more clearly delineate functionally conserved segments of untranslated regions. The near future should see the completion of many additional drosophilid and vertebrate species, which will provide an incredible resource for all informatic studies of regulatory biology, including that of miRNAs.