Non-coding RNAs (ncRNAs) exist in many flavors and facilitate a variety of molecular processes, from X chromosome inactivation to splicing to translational regulation. In a recent paper by Kohtz and colleagues (Genes Dev. 20, 1470–1484, 2006), a ncRNA is directly implicated in gene regulation by homeodomain transcription factors during vertebrate brain patterning.

The authors isolated a non-coding transcript, evf-2, corresponding to an ultraconserved region (> 90% conserved from fish to humans) that lies between the homeodomain transcription factors Dlx-5 and Dlx-6. evf-2 is a novel splice form of evf-1, a non-coding RNA previously isolated from specific regions of the brain, and is a polyadenylated, single stranded RNA. Although the authors do not exclude the possibility of an evf-2–encoded peptide, the RNA contains few open reading frames, suggesting that it is indeed a ncRNA.

evf-2 expression is responsive to sonic hedgehog (shh), a signaling molecule that plays a major role in developmental patterning, as viral overexpression of shh in mouse forebrains results in increased evf-2 expression. In addition, expression of a reporter carrying the dlx-5/6 region is increased dose-dependently by the evf-2 RNA, in a fashion dependent upon the Dlx-2 homeodomain protein. This data is consistent with evf-2 acting together with Dlx-2 to regulate transcription.

Reporter-based experiments suggest that the effect of evf-2 on Dlx-2 transcription is specific to the dlx-5/6 enhancer and cell line specific, perhaps suggesting tissue-specificity. Moreover, the effect of evf-2 on gene expression is strongest in combination with Dlx-2; evf-2 has less effect on regulation by other Dlx proteins and little effect on other homeodomain proteins.

Further experiments using the dlx-5/6 reporter assay indicate that evf-2 does not repress known Dlx-2 inhibitors, so the possibility of a direct interaction between the ncRNA and the homeodomain protein was tested. Dlx-2 forms a complex with evf-2 in cells, and evf-2 can be detected in immunoprecipitates of Dlx-family proteins from embryonic nuclear extracts, suggesting an in vivo interaction. In addition fluorescent in situ hybridization detects two evf-2 foci that colocalize with Dlx-2 in cell nuclei within a specific region of the developing mouse forebrain (see picture). While further experiments are required, these data tantalizingly suggest a direct interaction between the two factors.

It is known that fly Rox RNAs upregulate male X chromosome transcription, while the SRA ncRNA increases steroid receptor gene transcription. Further work will reveal whether there is a common mechanistic basis for ncRNA activity in these different systems and whether other genomic regions contain ncRNAs that regulate transcription of nearby genes.