A new class of non-coding RNAs associated with 3\u2019 untranslated regions of mRNAs

The importance of non-coding RNAs (ncRNAs) in controlling gene expression is becoming increasingly evident. However, except for some well characterized examples, such as miRNAs, Xist and Air, the function of most non-coding transcripts is still to be determined. Moreover, while small regulatory RNAs can be relatively easily classified on the basis of their length, secondary structure, and biochemical pathway, the classification of long \u201cmRNA-like\u201d ncRNAs has been problematic. Here we identify a large class of non-coding transcripts that originate within the 3\u2019UTR of at least one third of all genes in the mouse genome. We have several lines of evidence from genome-wide bioinformatic analyses (EST coverage, CAGE data, chromatin state maps of active promoters) and from invitro studies (strand-specific RT-PCR, 5\u2019RACE, Northern blot) showing that these 3\u2019UTR-associated ncRNAs (uaRNAs) can be either linked or transcribed separately to the upstream protein-coding sequences. In addition, expression profiles obtained by custom-designed microarrays on three different developmental systems (myoblast differentiation, male gonadal ridge formation, embryonic stem cell differentiation) showed that uaRNA expression is highly regulated and tissue-specific, and might be either concordant or discordant with respect to the upstream coding region depending on the cell type and on the developmental stage. This observation is confirmed by in-situ hybridization experiments, which evidenced that uaRNA and the associated coding transcript might have different subcellular locations. Our results highlight a further level of complexity at 3\u2019UTRs, suggesting the presence of new regulatory mechanisms that control gene expression during embryonic development. Our data have also important implications for the design of in-situ hybridization and microarray probes as well as for the interpretation of gene expression dat

Regulated independent expression of 3' untranslated regions in mammals

Eukaryotic mRNA localization, translation and stability are regulated by 3' untranslated regions (3'UTRs). 3'UTRs control mRNA expression in cis, via regulatory elements that are recognized by trans-acting factors, including RNA-binding proteins and microRNAs. However, a recent analysis of transcription start sites in mouse suggested that 3'UTRs may also be the source of independent transcripts, but there have been no in vivo analyses to confirm this suggestion. Here we report that a large number of 3'UTRs are not only linked to but are also expressed separately from their associated protein-coding sequences. We identify ~14,000 mouse genes exhibiting independent expression of 3'UTRs, of which 1,385 were supported by two distinct experimental approaches, and ~100 confirmed by microarray expression profiles. In situ hybridization demonstrated that 3'UTR-associated transcripts (uaRNA) can be expressed as part of mRNAs or discordantly in a developmentally-regulated and cell-specific manner, in some cases localizing to the nucleus. We also provide evidence that chromatin remodelling and transcription factor networks are involved in regulating uaRNA expression. These observations suggest that 3'UTRs not only function in cis to regulate protein expression but also in trans as noncoding RNAs, a conclusion supported by genetic studies dating back over a decade. Our findings prompt a re-evaluation of 3'UTR biology and the complexity of genome architecture in mammals

Characterization of long noncoding RNAs associated with developmental genes in vertebrates

The past few years have revealed that the genome of all studied organisms is almost entirely transcribed, generating an enormous number of non-protein-coding RNAs (ncRNAs) that are often organised in interlacing patterns with protein-coding genes. However, the functions of these RNAs remain largely untested. To identify target ncRNAs for characterisation, we analysed the expression of ncRNAs associated with key regulatory genes using mouse embryonic stem (ES) cells as a model of early development. Using a custom-designed microarray, we detected hundreds of ncRNAs whose expression is dynamically regulated during ES cell differentiation over a 16-day time course. The expression profile of many ncRNAs was either positively or negatively correlated with the expression of associated protein-coding genes with important roles in ES cell pluripotency and differentiation, such as Sox2, GATA6 and several homeobox-containing genes. We found that different isoforms of noncoding Sox2 Overlapping Transcripts (Sox2OT) transcribed from highly conserved elements have complex spatio-temporal relationships with Sox2 expression. By RT-PCR, whole-mount and brain section in situ hybridisations, we observed specific, and developmentally regulated, expression of Sox2OT transcripts in zebrafish, chicken and mouse embryos. We also identified novel spliced antisense (AS) ncRNAs that share similar expression patterns with associated homeotic genes, including Evx1,HoxB5/6 and all three Dlx clusters. We show that some of these ncRNA-mRNA pairs are spatially co-expressed in the mouse embryo and this co-regulation is maintained in adult tissues. In addition, we show that Evx1AS and HoxB5/6AS ncRNAs co-immunoprecipitate with trimethylated H3K4 histones and trithorax Mll1 chromatin fractions, involved in homeotic gene activation. These results suggest a positive role for ncRNAs in epigenetic regulation and homeotic programming. Taken together the data indicates that long ncRNAs participate in processes directing vertebrate developmental programs