In ovo application of antagomiRs indicates a role for miR-196 in patterning the chick axial skeleton through Hox gene regulation.

Patterning of the vertebrate axial skeleton requires precise spatial and temporal control of Hox gene expression during embryonic development. MicroRNAs (miRNAs) are recently described modulators of gene activity, and members of the miR-196 and miR-10 families have been shown to target several Hox genes in vivo. Testing miRNA function in mice is complicated by potential redundancy between family members. To circumvent this, we have developed protocols for introducing modified antisense oligonucleotides (antagomiRs) in ovo during chick development. Using this approach, we identify a layer of regulatory control provided by the miR-196 family in defining the boundary of Hox gene expression along the anterior-posterior (A-P) embryonic axis. Following knockdown of miR-196, we observe a homeotic transformation of the last cervical vertebrae toward a thoracic identity. This phenotypic alteration is, in part, due to an anterior expansion of Hoxb8 gene expression and consolidates the in vivo relevance of post-transcriptional Hox gene regulation provided by miRNAs in the complex hierarchies governing axial pattering.

MicroRNAs in the Hox network: an apparent link to posterior prevalence.

Homeobox (Hox) transcription factors confer anterior-posterior (AP) axial coordinates to vertebrate embryos. Hox genes are found in clusters that also contain genes for microRNAs (miRNAs). Our analysis of predicted miRNA targets indicates that Hox cluster-embedded miRNAs preferentially target Hox mRNAs. Moreover, the presumed Hox target genes are predominantly situated on the 3' side of each Hox miRNA locus. These results suggest that Hox miRNAs help repress more anterior programmes, thereby reinforcing posterior prevalence, which is the hierarchical dominance of posterior over anterior Hox gene function that is observed in bilaterians. In this way, miRNA-mediated regulation seems to recapitulate interactions at other levels of gene expression, some more ancestral, within a network under stabilizing selection.

The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development.

MicroRNAs (miRNAs) are an abundant class of gene regulatory molecules (reviewed in refs 1, 2). Although computational work indicates that miRNAs repress more than a third of human genes, their roles in vertebrate development are only now beginning to be determined. Here we show that miR-196 acts upstream of Hoxb8 and Sonic hedgehog (Shh) in vivo in the context of limb development, thereby identifying a previously observed but uncharacterized inhibitory activity that operates specifically in the hindlimb. Our data indicate that miR-196 functions in a fail-safe mechanism to assure the fidelity of expression domains that are primarily regulated at the transcriptional level, supporting the idea that many vertebrate miRNAs may function as a secondary level of gene regulation.

Patterns of flanking sequence conservation and a characteristic upstream motif for microRNA gene identification.

MicroRNAs are approximately 22-nucleotide (nt) RNAs processed from foldback segments of endogenous transcripts. Some are known to play important gene regulatory roles during animal and plant development by pairing to the messages of protein-coding genes to direct the post-transcriptional repression of these messages. Previously, we developed a computational method called MiRscan, which scores features related to the foldbacks, and used this algorithm to identify new miRNA genes in the nematode Caenorhabditis elegans. In the present study, to identify sequences that might be involved in processing or transcriptional regulation of miRNAs, we aligned sequences upstream and downstream of orthologous nematode miRNA foldbacks. These alignments showed a pronounced peak in sequence conservation about 200 bp upstream of the miRNA foldback and revealed a highly significant sequence motif, with consensus CTCCGCCC, that is present upstream of almost all independently transcribed nematode miRNA genes. Scoring the pattern of upstream/downstream conservation, the occurrence of this sequence motif, and orthology of host genes for intronic miRNA candidates, yielded substantial improvements in the accuracy of MiRscan. Nine new C. elegans miRNA gene candidates were validated using a PCR-sequencing protocol. As previously seen for bacterial RNA genes, sequence features outside of the RNA secondary structure can therefore be very useful for the computational identification of eukaryotic noncoding RNA genes. The total number of confidently identified nematode miRNAs now approaches 100. The improved analysis supports our previous assertion that miRNA gene identification is nearing completion in C. elegans with apparently no more than 20 miRNA genes now remaining to be identified.

MicroRNA-directed cleavage of HOXB8 mRNA.

MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs, some of which are known to play important regulatory roles in animals by targeting the messages of protein-coding genes for translational repression. We find that miR-196, a miRNA encoded at three paralogous locations in the A, B, and C mammalian HOX clusters, has extensive, evolutionarily conserved complementarity to messages of HOXB8, HOXC8, and HOXD8. RNA fragments diagnostic of miR-196-directed cleavage of HOXB8 were detected in mouse embryos. Cell culture experiments demonstrated down-regulation of HOXB8, HOXC8, HOXD8, and HOXA7 and supported the cleavage mechanism for miR-196-directed repression of HOXB8. These results point to a miRNA-mediated mechanism for the posttranscriptional restriction of HOX gene expression during vertebrate development and demonstrate that metazoan miRNAs can repress expression of their natural targets through mRNA cleavage in addition to inhibiting productive translation.

The microRNAs of Caenorhabditis elegans.

MicroRNAs (miRNAs) are an abundant class of tiny RNAs thought to regulate the expression of protein-coding genes in plants and animals. In the present study, we describe a computational procedure to identify miRNA genes conserved in more than one genome. Applying this program, known as MiRscan, together with molecular identification and validation methods, we have identified most of the miRNA genes in the nematode Caenorhabditis elegans. The total number of validated miRNA genes stands at 88, with no more than 35 genes remaining to be detected or validated. These 88 miRNA genes represent 48 gene families; 46 of these families (comprising 86 of the 88 genes) are conserved in Caenorhabditis briggsae, and 22 families are conserved in humans. More than a third of the worm miRNAs, including newly identified members of the lin-4 and let-7 gene families, are differentially expressed during larval development, suggesting a role for these miRNAs in mediating larval developmental transitions. Most are present at very high steady-state levels-more than 1000 molecules per cell, with some exceeding 50,000 molecules per cell. Our census of the worm miRNAs and their expression patterns helps define this class of noncoding RNAs, lays the groundwork for functional studies, and provides the tools for more comprehensive analyses of miRNA genes in other species.

The contribution of 2'-hydroxyls to the cleavage activity of the Neurospora VS ribozyme.

We have used nucleotide analog interference mapping and site-specific substitution to determine the effect of 2'-deoxynucleotide substitution of each nucleotide in the VS ribozyme on the self-cleavage reaction. A large number of 2'-hydroxyls (2'-OHs) that contribute to cleavage activity of the VS ribozyme were found distributed throughout the core of the ribozyme. The locations of these 2'-OHs in the context of a recently developed helical orientation model of the VS ribozyme suggest roles in multi-stem junction structure, helix packing, internal loop structure and catalysis. The functional importance of three separate 2'-OHs supports the proposal that three uridine turns contribute to local and long-range tertiary structure formation. A cluster of important 2'-OHs near the loop that is the candidate region for the active site and one very important 2'-OH in the loop that contains the cleavage site confirm the functional importance of these two loops. A cluster of important 2'-OHs lining the minor groove of stem-loop I and helix II suggests that these regions of the backbone may play an important role in positioning helices in the active structure of the ribozyme.