754 VOLUME 13 NUMBER 9 SEPTEMBER 2006 NATURE STRUCTURAL & MOLECULAR BIOLOGY

The author is at the Max Delbrück Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, Berlin-Buch, 13092 Berlin, Germany.e-mail: rajewsky@mdc-berlin.de

L(ou)sy miRNA targets?Nikolaus Rajewsky

MicroRNAs (miRNAs) are noncoding small RNAs thought to post-transcriptionally regulate many metazoan genes by binding to partially complementary sites in target messenger RNAs. In this issue, Didiano and Hobert examine known and predicted targets of the nematode lsy-6 miRNA, question the general validity of previously proposed rules about miRNA-target interactions and suggest that many functional miRNA targets might be context dependent, as seen in metazoan gene regulation by transcription factors.

In the past few years, it has been demonstrated that a large class of noncoding small RNAs (miRNAs) exists in metazoa and plants, that they post-transcriptionally regulate a large fraction of all genes and that they are important regulators of a broad spectrum of biological processes, including morphogenesis, signaling, metabolism and cancer. Animal miRNAs can bind partially complementary target sites in the 3′ untranslated regions (UTRs) of mRNAs and, by poorly understood mechanisms, repress translation, alter mRNA stability or induce deadenylation of their targets (for reviews, see refs. 1 and 2). To understand the specific biological function of an miRNA, it is therefore crucial to identify its specific target mRNAs. A steeply growing number of recent experimental and computational studies have focused on this problem, and their results strongly suggest that phylogenetically conserved sites containing 6 to 8 base pairs (bp) of consecutive Watson-Crick matches to the 5′ region of an miRNA (‘seed matches’) are reasonable predictors of at least one major class of its functional targets. However, many (but not all) of the in vivo experimental systems that have been designed to systematically assay miRNA- target interactions have used overexpression of miRNAs, misexpression of the targets or both, thus potentially generating results that are not congruent with endogenous miRNA-target interactions.

Previous studies from the Hobert lab have pioneered the elucidation of the gene regulatory network that establishes asymmetry between two morphologically bilateral taste- receptor neurons in the nema-tode Caenorhabditis elegans, termed ASEL and ASER (the ‘left’ and ‘right’ chemosensory neu-rons, respectively), which enable C. elegans to respond to different chemosensory cues (for a review, see ref. 3). A nematode miRNA termed lousy-6 (lsy-6) turned out to be one of the key factors in establishing left-right asymmetry between ASEL and ASER. An essential feature of the regulatory network responsible for the entire developmental process is that lsy-6, expressed in ASEL but not in ASER, directly represses the transcription factor gene cog-1, which is active in ASER.

To test computationally predicted lsy-6 miRNA targets and to further examine the already established lsy-6 target site in the cog-1 3′ UTR, a new study by Didiano and Hobert4 on page 849 of this issue uses an in vivo sensor system developed by the Hobert lab over the past few years. A reporter gene (green fluor-escent protein, GFP) is fused to the 3′ UTR of interest and its expression driven by a heterologous promoter in both ASEL and ASER. This in vivo system has the advantage that the endogenous miRNA expression level is not directly altered. Further, to counter the inherently variable effects of extrachromo-somal DNA arrays, GFP levels are directly compared between two single cells (ASEL versus ASER) within one animal. The repro-ducibility of the differences is then assayed by observing two or three transgenic lines. In these experiments, downregulation of the

GFP sensor construct in ASEL compared to ASER indicates a lsy-6 target.

From this assay, Didiano and Hobert report four important observations. First, the one established functional lsy-6 target site in the cog-1 3′ UTR can tolerate non–Watson-Crick base pairs (G•U ‘wobbles’) at various posi-tions in its ‘seed region’ (Fig. 1, blue). Second, although the seed is required for cog-1 repres-sion by lsy-6, only the entire site is sufficient to repress cog-1. Third, the complete lsy-6 target site in cog-1 can induce downregulation of lin-28, but not unc-54, when fused into their respective 3′ UTRs. Fourth, none of 13 compu-tationally predicted, conserved lsy-6 target sites in 13 different 3′ UTRs are regulated by lsy-6.

On the basis of these results, Didiano and Hobert conclude that (i) G•U wobbles in the seed region of an miRNA bound to a target may be tolerated, (ii) a conserved, predicted target site for lsy-6 is not necessarily regulated by this miRNA in ASEL, and (iii) target sites may need a certain 3′ UTR context to be functional. In conclusions (ii) and (iii), one might replace ‘target site’ with ‘transcription factor–binding site’, ‘3′ UTR’ with ‘promoter’ or ‘enhancer’, and ‘miRNA’ with ‘transcription factor’. Thus, there may be general conceptual similarities between post-transcriptional gene regulation mediated by miRNAs and well-known mechanisms for enhancing the specificity of transcriptional gene regulation in eukaryotes. In particular, a single metazoan transcription factor bound to its cognate binding site is, very often, func-tional only in conjunction with additional (nearby) bound transcription factors or other cofactors, or only in certain chromatin states (see ref. 3 and references therein). In analogy,

N E W S A N D V I E W S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

smb

NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 13 NUMBER 9 SEPTEMBER 2006 755

it is conceivable that the functionality of an miRNA-binding site depends not only on the presence of the cognate miRNA but also on the presence nearby of other binding sites and their cognate factors, such as miRNAs and RNA-binding proteins, as well as on the secondary structure of the target mRNA. For example, an RNA-binding protein specifically expressed in a certain cellular context could prevent an miRNA from binding its cognate binding site. It seems worth pointing out here that each metazoan genome contains hundreds to thou-sands of proteins with RNA-binding domains, most of them with unknown function.

Before I discuss the general overall implications of this study, some open ques-tions remain. The cog-1 3′ UTR contains another conserved, predicted target site for lsy-6 (Fig. 1, yellow). This site is rela-tively close to the site assayed by Didiano and Hobert, and it is possible that the two sites interact with each other. Interestingly, the well-known regulation of lin-41 by the miRNA let-7 would resemble this. Both cog-1 and lin-41 have been identified as strong genetic targets of the respective miRNAs, and the lin-41 3′ UTR contains two nearby noncanonical let-7 target sites that prob-ably interact with each other, as the spacer between the noncanonical sites is required for their functionality (see ref. 5 and refer-ences therein). Therefore, even if the second predicted site in the 3′ UTR of cog-1 is not required for cog-1 regulation by lsy-6, it may not be appropriate to infer or disprove rules

about single–target site recognition by lsy-6 from the results of mutations in the first site, without reference to the second. For example, it is conceivable that, upon removal of the sec-ond site, G•U wobbles would be detrimental to the functionality of the first site.

Another open question concerns the use of a heterologous promoter in the sensor assay. Although the positive control (cog-1) works properly in this experiment, it is not clear whether lsy-6 regulation of the 13 other pre-dicted lsy-6 targets tested would be detected in this assay, as their mRNA concentrations almost certainly do not correspond to endo-genous levels. The experimental design may, for example, alter relative concentrations of lsy-6 targets and thus, indirectly, the effective concentration of lsy-6 itself.

However, based on this assay, how can we explain one of the main findings of Didiano and Hobert, that none of the 13 predicted tar-gets seems to be functional in ASEL? First, it could be that lsy-6 is simply an unusual miRNA that recognizes targets differently than most other miRNAs. However, this seems unlikely, as the target predictions correctly (and with high scores) identify cog-1. Second, it could be that lsy-6 is a perfectly normal miRNA and that the target predictions are wrong. This interpreta-tion, however, conflicts with a growing volume of evidence that target predictions based on conserved seed sites work well (see ref. 2 and references therein). Third, as the authors point out, it seems that, at least for lsy-6, conserved seed sites cannot necessarily be expected to

mediate repression of their targets in a given cell (ASEL) where a given miRNA (lsy-6) is expressed, but they could do so in other cells where lsy-6 is expressed or at other develop-mental stages. This interpretation potentially questions previous suggestions that many seed sites should be functional in the presence of the miRNA, regardless of 3′ UTR sequence con-text6. It also suggests that, to test the functions of predicted miRNA targets, it may be useful to employ reporter systems designed to assay the endogenous expression of these targets in the entire animal7. In sum, as the numbers are small (one miRNA in one cell), generalizations of any sort are dangerous, but the fascinating current study demonstrates that our under-standing of miRNA-target interactions is very limited. The study also opens up interesting possibilities, both experimental and computa-tional, to further investigate mechanisms that may contribute to context-dependent gene regulation mediated by miRNAs.

ACKNOWLEDGMENTSI would like to thank S. Small, F. Piano and K. Gunsalus for helpful discussions and F. Piano and K. Gunsalus for critical comments on the manuscript.

1. Bartel, D.P. Cell 116, 281–297 (2004).2. Rajewsky, N. Nat. Genet. 38, s8–13 (2006).3. Davidson, E. The Regulatory Genome: Gene Regulatory

Networks In Development And Evolution (Academic Press, Elsevier, 2006).

4. Didiano, D. & Hobert, O. Nat. Struct. Mol. Biol. 13, 849–851 (2006).

5. Vella, M.C., Reinert, K. & Slack, F.J. Chem. Biol. 11, 1619–1623 (2004).

6. Farh, K.K. et al. Science 310, 1817–1821 (2005).7. Lall, S. et al. Curr. Biol. 16, 460–471 (2006).

ATCGTCACTTATACAAAAACCAAACTCC--------CTTTTACCGTT---------------------ATCGTCACTTATACAAAA-------TCCCCCCAAAACTTCACCCGTTTTCTTTATTAAAAATGCGTCTATCGTCATTCATACAAAAACTTCTTTCCCCTCAAAA----ATCCGTTTCC-------AAAATGCGTC-

-----------AAACCATGCCCAAATACAA-----AAAATTTCCCA------TTTAATTGTACGTTTCCCATACAAAAAAATAACTCTTAAATACAAAATGTAAAATTTCTCATCCCTTTGTGAATGTATTTTTCCAATACAAAAACTT--CCCTAATATACAAAATGTA-------------CTTTTTCAATGTATTTTT

C. elegansC. briggsaeC. remanei

C. elegansC. briggsae

C. remanei

Figure 1 Multiple sequence alignment of the cog-1 3′ UTR and predicted lsy-6 target sites. Shown are sequences from three Caenorhabditis species, retrieved from http://pictar.bio.nyu.edu. Beginning and end of the 3′ UTR are truncated to improve readability. Colors highlight parts of predicted lsy-6 target sites that are complementary to the 5′ end of lsy-6 (seed regions). Site assayed by Didiano and Hobert4 is in blue.

N E W S A N D V I E W S©

2006

Nat

ure

Pub

lishi

ng G

roup

ht

tp://

ww

w.n

atur

e.co

m/n

smb